Publications by authors named "Guido Kroemer"

1,069 Publications

  • Page 1 of 1

Cell death in pancreatic cancer: from pathogenesis to therapy.

Nat Rev Gastroenterol Hepatol 2021 Jul 30. Epub 2021 Jul 30.

Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The Third Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China.

Pancreatic cancer is a devastating gastrointestinal cancer characterized by late diagnosis, limited treatment success and dismal prognosis. Exocrine tumours account for 95% of pancreatic cancers and the most common pathological type is pancreatic ductal adenocarcinoma (PDAC). The occurrence and progression of PDAC involve multiple factors, including internal genetic alterations and external inflammatory stimuli. The biology and therapeutic response of PDAC are further shaped by various forms of regulated cell death, such as apoptosis, necroptosis, ferroptosis, pyroptosis and alkaliptosis. Cell death induced by local or systemic treatments suppresses tumour proliferation, invasion and metastasis. However, unrestricted cell death or tissue damage might result in an inflammation-related immunosuppressive microenvironment, which is conducive to tumour progression or recurrence. The precise extent to which cell death affects PDAC is not yet well described. A growing body of preclinical and clinical studies document significant correlations between mutations (for example, in KRAS and TP53), stress responses (such as hypoxia and autophagy), metabolic reprogramming and chemotherapeutic responses. Here, we describe the molecular machinery of cell death, discuss the complexity and multifaceted nature of lethal signalling in PDAC cells, and highlight the challenges and opportunities for activating cell death pathways through precision oncology treatments.
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http://dx.doi.org/10.1038/s41575-021-00486-6DOI Listing
July 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

Immunoblot-based assays for assessing autophagy in the turquoise killifish Nothobranchius furzeri.

Methods Cell Biol 2021 18;165:123-138. Epub 2020 Nov 18.

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; 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:

Autophagy is an evolutionarily conserved biological process required for the turnover of the cytoplasm of eukaryotic cell. Beyond its catabolic nature, autophagy has a plethora of pro-survival functions, thus combatting hypoxia, nutrient shortage, and unfolded protein accumulation. Here, we introduce the naturally short-lived turquoise killifish Nothobranchius furzeri as an emerging model to study autophagic function in vivo, in response to environmental challenges. We show that starvation in killifish is sufficient to increase autophagic flux in the liver, thus enhancing the lipidation of microtubule-associated protein light chain 3 (LC3) and reducing the abundance of the autophagic substrate sequestosome-1 (SQSTM1). We describe an immunoblot-based comprehensive protocol to monitor fluctuations in autophagy in this model organism.
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http://dx.doi.org/10.1016/bs.mcb.2020.10.007DOI Listing
November 2020

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

Elevated plasma levels of the appetite-stimulator ACBP/DBI in fasting and obese subjects.

Cell Stress 2021 Jul 28;5(7):89-98. Epub 2021 Jun 28.

Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Inserm U1138, Université de Paris, Sorbonne Université, Paris, France.

Eukaryotic cells release the phylogenetically ancient protein acyl coenzyme A binding protein (ACBP, which in humans is encoded by the gene DBI, diazepam binding inhibitor) upon nutrient deprivation. Accordingly, mice that are starved for one to two days and humans that undergo voluntary fasting for one to three weeks manifest an increase in the plasma concentration of ACBP/DBI. Paradoxically, ACBP/DBI levels also increase in obese mice and humans. Since ACBP/DBI stimulates appetite, this latter finding may explain why obesity constitutes a self-perpetuating state. Here, we present a theoretical framework to embed these findings in the mechanisms of weight control, as well as a bioinformatics analysis showing that, irrespective of the human cell or tissue type, one single isoform of ACBP/DBI (ACBP1) is preponderant (~90% of all DBI transcripts, with the sole exception of the testis, where it is ~70%). Based on our knowledge, we conclude that ACBP1 is subjected to a biphasic transcriptional and post-transcriptional regulation, explaining why obesity and fasting both are associated with increased circulating ACBP1 protein levels.
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http://dx.doi.org/10.15698/cst2021.07.252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283301PMC
July 2021

Actinomycin D targets NPM1c-primed mitochondria to restore PML-driven senescence in AML therapy.

Cancer Discov 2021 Jul 23. Epub 2021 Jul 23.

The Campbell Family Institute for Breast Cancer Research, Princess Margaret Hospital, University of Toronto.

Acute myeloid leukemia (AML) pathogenesis often involves a mutation in the NPM1 nucleolar chaperone, but the bases for its transforming properties and overall association with favorable therapeutic responses remain incompletely understood. Here we demonstrate that an oncogenic mutant form of NPM1 (NPM1c) impairs mitochondrial function. NPM1c also hampers formation of PML nuclear bodies (NBs), which are regulators of mitochondrial fitness and key senescence effectors. Actinomycin D (ActD), an antibiotic with unambiguous clinical efficacy in relapsed/refractory NPM1c-AMLs, targets these primed mitochondria, releasing mtDNA, activating cGAS signaling and boosting ROS production. The latter restore PML NB formation to drive TP53 activation and senescence of NPM1c-AML cells. In several models, dual targeting of mitochondria by venetoclax and ActD synergized to clear AML and prolong survival through targeting of PML. Our studies reveal an unexpected role for mitochondria downstream of NPM1c and implicate a mitochondrial/ROS/PML/TP53 senescence pathway as an effector of ActD-based therapies.
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http://dx.doi.org/10.1158/2159-8290.CD-21-0177DOI Listing
July 2021

Pyridoxal kinase and poly(ADP-ribose) affect the immune microenvironment of locally advanced cancers.

Oncoimmunology 2021 8;10(1):1950954. Epub 2021 Jul 8.

Equipe 11 labellisée par la Ligue contre le Cancer, Université de Paris, Sorbonne Université, INSERM U1138, Centre de Recherche des Cordeliers, Paris, France.

Malignant cells adapt to the hostile tumor microenvironment by escaping from, or actively suppressing, anticancer immune responses. In the past, we reported that reduced synthesis of active vitamin B6 (due to downregulation of pyridoxal kinase) or overactivation of poly(ADP-ribose) polymerase confers resistance to chemotherapy with cisplatin. Recently, we found that these prognostically adverse alterations in oncometabolism also correlate with the rarefaction of immune effectors in the tumor bed.
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http://dx.doi.org/10.1080/2162402X.2021.1950954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8274443PMC
August 2021

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

Cell Death Dis 2021 Jul 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 Anti-Tumor Responses In Vivo.

Mol Cancer Ther 2021 Jul 12. Epub 2021 Jul 12.

Oncology R&D, GlaxoSmithKline (United States).

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma (MM) and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug-conjugate approved for the treatment of relapsed/refractory MM. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo. GSK2857916 treatment enhances intra-tumor 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 re-challenge 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 anti-tumor 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
July 2021

The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma.

Blood 2021 07 7. Epub 2021 Jul 7.

Université de Paris, NF-kappaB, Différenciation et Cancer, Paris, France

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. NF-kB transcription factor family is activated by two main pathways, the canonical and the alternative NF-kB activation pathways with different functions. The alternative NF-kB pathway leads to the activation of the transcriptionally active RelB NF-kB subunit. Alternative NF-kB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their ABC or GCB subtypes. RelB activity defines a new subset of DLBCL patients with a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for ABC tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-kB, thus indicating that current genetic tools to evaluate NF-kB activity in DLBCL do not provide information on the alternative NF-kB activation. Further, the newly defined RelB-positive subgroup of DLBCL patients exhibits a dismal outcome following immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA-damage induced apoptosis in response to doxorubicin, a genotoxic agent used in front-line treatment for DLBCL. We also show that RelB positivity is associated with high expression of cIAP2. Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of DLBCL patients.
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http://dx.doi.org/10.1182/blood.2020010039DOI Listing
July 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

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

Metabolic features of cancer cells impact immunosurveillance.

J Immunother Cancer 2021 Jun;9(6)

Equipe 11 labellisée par la Ligue contre le Cancer, Université de Paris, Sorbonne Université, INSERM U1138, Centre de Recherche des Cordeliers, INSERM, Paris, France

Background: Tumors rewire their metabolism to achieve robust anabolism and resistance against therapeutic interventions like cisplatin treatment. For example, a prolonged exposure to cisplatin causes downregulation of pyridoxal kinase (PDXK), the enzyme that generates the active vitamin B6, and upregulation of poly ADP-ribose (PAR) polymerase-1 (PARP1) activity that requires a supply of nicotinamide (vitamin B3) adenine dinucleotide. We investigated the impact of the levels of PDXK and PAR on the local immunosurveillance (ie, density of the antigen presenting cells and adaptive immune response by CD8 T lymphocytes) in two different tumor types.

Methods: Tumors from patients with locally advanced cervical carcinoma (LACC) and non-small cell lung cancer (NSCLC) were stained for PAR, PDXK, dendritic cell lysosomal associated membrane glycoprotein (DC-LAMP) and CD8 T cell infiltration. Their correlations and prognostic impact were assessed. Cisplatin-resistant NSCLC cell clones isolated from Lewis-lung cancer (LLC) cells were evaluated for PAR levels by immunoblot. Parental (PAR) and cisplatin-resistant (PAR) clones were subcutaneously injected into the flank of C57BL/6 mice. Tumors were harvested to evaluate their immune infiltration by flow cytometry.

Results: The infiltration of tumors by CD8 T and DC-LAMP cells was associated with a favorable overall survival in patients with LACC (p=0.006 and p=0.008, respectively) and NSCLC (p<0.001 for both CD8 T and DC-LAMP cells). We observed a positive correlation between PDXK expression and the infiltration by DC-LAMP (R=0.44, p=0.02 in LACC, R=0.14, p=0.057 in NSCLC), and a negative correlation between PAR levels and CD8 T lymphocytes (R=-0.39, p=0.034 in LACC, R=-0.18, p=0.017 in NSCLC). PARP1 is constitutively hyperactivated in cisplatin-resistant LLC cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PAR). Tumors formed by such cancer cells injected into immunocompetent mice were scarcely infiltrated by CD8 T (p=0.028) and antigen presenting cells (p=0.086).

Conclusions: Oncometabolic features can impact local immunosurveillance, providing new functional links between cisplatin resistance and therapeutic failure.
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http://dx.doi.org/10.1136/jitc-2021-002362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8231002PMC
June 2021

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

J Immunother Cancer 2021 Jun;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 Jun 10:1-2. 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
June 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

Effects of acyl-coenzyme A binding protein (ACBP)/diazepam-binding inhibitor (DBI) on body mass index.

Cell Death Dis 2021 Jun 9;12(6):599. Epub 2021 Jun 9.

Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Inserm U1138, Université de Paris, Sorbonne Université, Paris, France.

In mice, the plasma concentrations of the appetite-stimulatory and autophagy-inhibitory factor acyl-coenzyme A binding protein (ACBP, also called diazepam-binding inhibitor, DBI) acutely increase in response to starvation, but also do so upon chronic overnutrition leading to obesity. Here, we show that knockout of Acbp/Dbi in adipose tissue is sufficient to prevent high-fat diet-induced weight gain in mice. We investigated ACBP/DBI plasma concentrations in several patient cohorts to discover a similar dual pattern of regulation. In relatively healthy subjects, ACBP/DBI concentrations independently correlated with body mass index (BMI) and age. The association between ACBP/DBI and BMI was lost in subjects that underwent major weight gain in the subsequent 3-9 years, as well as in advanced cancer patients. Voluntary fasting, undernutrition in the context of advanced cancer, as well as chemotherapy were associated with an increase in circulating ACBP/DBI levels. Altogether, these results support the conclusion that ACBP/DBI may play an important role in body mass homeostasis as well as in its failure.
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http://dx.doi.org/10.1038/s41419-021-03864-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190068PMC
June 2021

Spermidine-induced hypusination preserves mitochondrial and cognitive function during aging.

Autophagy 2021 Jun 9:1-3. Epub 2021 Jun 9.

Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria.

Spermidine is a natural polyamine, central to cellular homeostasis and growth, that promotes macroautophagy/autophagy. The polyamine pathway is highly conserved from bacteria to mammals and spermidine (prominently found in some kinds of aged cheese, wheat germs, nuts, soybeans, and fermented products thereof, among others) is an intrinsic part of the human diet. Apart from nutrition, spermidine is available to mammalian organisms from intracellular biosynthesis and microbial production in the gut. Importantly, externally supplied spermidine (via drinking water or food) prolongs lifespan, activates autophagy, improves mitochondrial function, and refills polyamine pools that decline during aging in various tissues of model organisms, including mice. In two adjacent studies, we explored how dietary spermidine supplementation enhances eEF5/EIF5A hypusination, cerebral mitochondrial function and cognition in aging and mice.
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http://dx.doi.org/10.1080/15548627.2021.1933299DOI Listing
June 2021

Autophagy in metabolism and quality control: opposing, complementary or interlinked functions?

Autophagy 2021 Jun 22:1-10. Epub 2021 Jun 22.

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 autophagy, macroautophagy, is considered to be both a metabolic process as well as a bona fide quality control process. The question as to how these two aspects of autophagy are coordinated and whether and why they overlap has implications for fundamental aspects, pathophysiological effects, and pharmacological manipulation of autophagy. At the top of the regulatory cascade controlling autophagy are master regulators of cellular metabolism, such as MTOR and AMPK, which render the system responsive to amino acid and glucose starvation. At the other end exists a variety of specific autophagy receptors, engaged in the selective removal of a diverse array of intracellular targets, from protein aggregates/condensates to whole organelles such as mitochondria, ER, peroxisomes, lysosomes and lipid droplets. Are the roles of autophagy in metabolism and quality control mutually exclusive, independent or interlocked? How are priorities established? What are the molecular links between both phenomena? This article will provide a starting point to formulate these questions, the responses to which should be taken into consideration in future autophagy-based interventions.
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http://dx.doi.org/10.1080/15548627.2021.1933742DOI Listing
June 2021

Targeting ferroptosis in pancreatic cancer: a double-edged sword.

Trends Cancer 2021 May 19. Epub 2021 May 19.

Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The Third Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China; Department of Surgery, University of Texas Southwestern Medical Center, Dallas, TX, USA. Electronic address:

Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy with a 5-year survival rate below 10%. Its unique genetic makeup and tumor microenvironment produce a lack of response to current treatments, including chemotherapy, radiotherapy, and immunotherapy. Recent preclinical studies have revealed that ferroptosis, an iron-dependent form of nonapoptotic cell death driven by unrestricted lipid peroxidation, may be an attractive therapeutic goal in PDAC. Understanding the dual role of ferroptotic cell death in both promoting and suppressing tumor immunity, as well as its integrated regulatory mechanisms and signaling pathways, may lead to more effective treatment designs for clinical trials of PDAC and may minimize or delay the emergence of drug resistance or side effects.
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http://dx.doi.org/10.1016/j.trecan.2021.04.005DOI Listing
May 2021

Ferritinophagy and ferroptosis in the management of metabolic diseases.

Trends Endocrinol Metab 2021 07 15;32(7):444-462. Epub 2021 May 15.

Shanghai Institute of Cardiovascular Diseases, Department of Cardiology, Zhongshan Hospital Fudan University, Shanghai 200032, China; Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA. Electronic address:

Ferroptosis is a form of regulated cell death modality associated with disturbed iron-homeostasis and unrestricted lipid peroxidation. Ample evidence has depicted an essential role for ferroptosis as either the cause or consequence for human diseases, denoting the likely therapeutic promises for targeting ferroptosis in the preservation of human health. Ferritinophagy, a selective form of autophagy, contributes to the initiation of ferroptosis through degradation of ferritin, which triggers labile iron overload (IO), lipid peroxidation, membrane damage, and cell death. In this review, we will delineate the role of ferritinophagy in ferroptosis, and its underlying regulatory mechanisms, to unveil the therapeutic value of ferritinophagy as a target in the combat of ferroptosis to manage metabolic diseases.
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http://dx.doi.org/10.1016/j.tem.2021.04.010DOI Listing
July 2021

A bacterium-derived, cancer-associated immunopeptidome.

Oncoimmunology 2021 05 3;10(1):1918373. Epub 2021 May 3.

Karolinska Institute, Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden.

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http://dx.doi.org/10.1080/2162402X.2021.1918373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096325PMC
May 2021

Hallmarks of T cell aging.

Nat Immunol 2021 06 13;22(6):687-698. Epub 2021 May 13.

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 aged adaptive immune system is characterized by progressive dysfunction as well as increased autoimmunity. This decline is responsible for elevated susceptibility to infection and cancer, as well as decreased vaccination efficacy. Recent evidence indicates that CD4 T cell-intrinsic alteratins contribute to chronic inflammation and are sufficient to accelerate an organism-wide aging phenotype, supporting the idea that T cell aging plays a major role in body-wide deterioration. In this Review, we propose ten molecular hallmarks to represent common denominators of T cell aging. These hallmarks are grouped into four primary hallmarks (thymic involution, mitochondrial dysfunction, genetic and epigenetic alterations, and loss of proteostasis) and four secondary hallmarks (reduction of the TCR repertoire, naive-memory imbalance, T cell senescence, and lack of effector plasticity), and together they explain the manifestation of the two integrative hallmarks (immunodeficiency and inflammaging). A major challenge now is weighing the relative impact of these hallmarks on T cell aging and understanding their interconnections, with the final goal of defining molecular targets for interventions in the aging process.
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http://dx.doi.org/10.1038/s41590-021-00927-zDOI Listing
June 2021

Ferroptosis in infection, inflammation, and immunity.

J Exp Med 2021 Jun 12;218(6). Epub 2021 May 12.

Guangzhou Municipal and Guangdong Provincial Key Laboratory of Protein Modification and Degradation, The Third Affiliated Hospital, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, China.

Ferroptosis is a type of regulated necrosis that is triggered by a combination of iron toxicity, lipid peroxidation, and plasma membrane damage. The upstream inducers of ferroptosis can be divided into two categories (biological versus chemical) and activate two major pathways (the extrinsic/transporter versus the intrinsic/enzymatic pathways). Excessive or deficient ferroptotic cell death is implicated in a growing list of physiological and pathophysiological processes, coupled to a dysregulated immune response. This review focuses on new discoveries related to how ferroptotic cells and their spilled contents shape innate and adaptive immunity in health and disease. Understanding the immunological characteristics and activity of ferroptotic death not only illuminates an intersection between cell death and immunity but may also lead to the development of novel treatment approaches for immunopathological diseases.
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http://dx.doi.org/10.1084/jem.20210518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126980PMC
June 2021
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