Publications by authors named "Raphaël Rodriguez"

65 Publications

Loss of SDHB Promotes Dysregulated Iron Homeostasis, Oxidative Stress, and Sensitivity to Ascorbate.

Cancer Res 2021 Jul 14;81(13):3480-3494. Epub 2021 Jun 14.

PARCC, INSERM UMR970, Equipe Labellisée par la Ligue Contre le Cancer, Paris, France.

Succinate dehydrogenase is a key enzyme in the tricarboxylic acid cycle and the electron transport chain. All four subunits of succinate dehydrogenase are tumor suppressor genes predisposing to paraganglioma, but only mutations in the SDHB subunit are associated with increased risk of metastasis. Here we generated an knockout chromaffin cell line and compared it with deficient cells. Both cell types exhibited similar SDH loss of function, metabolic adaptation, and succinate accumulation. In contrast, cells showed hallmarks of mesenchymal transition associated with increased DNA hypermethylation and a stronger pseudo-hypoxic phenotype compared with cells. Loss of SDHB specifically led to increased oxidative stress associated with dysregulated iron and copper homeostasis in the absence of NRF2 activation. High-dose ascorbate exacerbated the increase in mitochondrial reactive oxygen species, leading to cell death in cells. These data establish a mechanism linking oxidative stress to iron homeostasis that specifically occurs in -deficient cells and may promote metastasis. They also highlight high-dose ascorbate as a promising therapeutic strategy for SDHB-related cancers. SIGNIFICANCE: Loss of different succinate dehydrogenase subunits can lead to different cell and tumor phenotypes, linking stronger 2-OG-dependent dioxygenases inhibition, iron overload, and ROS accumulation following SDHB mutation.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2936DOI Listing
July 2021

Chemistry and biology of ferritin.

Metallomics 2021 05;13(5)

Chemical Biology of Cancer Laboratory, Institut Curie, 26 rue d'Ulm, 75005 Paris, France.

Iron is an essential element required by cells and has been described as a key player in ferroptosis. Ferritin operates as a fundamental iron storage protein in cells forming multimeric assemblies with crystalline iron cores. We discuss the latest findings on ferritin structure and activity and its link to cell metabolism and ferroptosis. The chemistry of iron, including its oxidation states, is important for its biological functions, its reactivity, and the biology of ferritin. Ferritin can be localized in different cellular compartments and secreted by cells with a variety of functions depending on its spatial context. Here, we discuss how cellular ferritin localization is tightly linked to its function in a tissue-specific manner, and how impairment of iron homeostasis is implicated in diseases, including cancer and coronavirus disease 2019. Ferritin is a potential biomarker and we discuss latest research where it has been employed for imaging purposes and drug delivery.
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http://dx.doi.org/10.1093/mtomcs/mfab021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083198PMC
May 2021

CD44 regulates epigenetic plasticity by mediating iron endocytosis.

Nat Chem 2020 10 3;12(10):929-938. Epub 2020 Aug 3.

Institut Curie, Paris, France.

CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.
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http://dx.doi.org/10.1038/s41557-020-0513-5DOI Listing
October 2020

Editorial overview: Toward smart medicines.

Curr Opin Chem Biol 2020 06 11;56:A1-A2. Epub 2020 May 11.

Chemical Biology of Cancer Laboratory, Institut Curie, CNRS, INSERM, PSL Université Paris, 75005 Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.cbpa.2020.04.011DOI Listing
June 2020

Salinomycin Derivatives Kill Breast Cancer Stem Cells by Lysosomal Iron Targeting.

Chemistry 2020 Jun 17;26(33):7416-7424. Epub 2020 Apr 17.

Institut Curie, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

Salinomycin (1) exhibits a large spectrum of biological activities including the capacity to selectively eradicate cancer stem cells (CSC), making it and its derivatives promising candidates for the development of drug leads against CSC. It has been previously shown that salinomycin and its C20-propargylamine derivative (Ironomycin (2)) accumulate in lysosomes and sequester iron in this organelle. Herein, a library of salinomycin derivatives is reported, including products of C20-amination, C1-esterification, C9-oxidation, and C28-dehydration. The biological activity of these compounds is evaluated against transformed human mammary epithelial HMLER CD24 /CD44 cells, a well-established model of breast CSC, and HMLER CD24 /CD44 cells deprived of CSC properties. Unlike other structural alterations, derivative 4, which displays a cyclopropylamine at position C20, showed a strikingly low IC value of 23 nm against HMLER CD24 /CD44 cells. This study provides highly selective molecules to target the CSC niche, a potential interesting advance for drug development to prevent cancer resistance.
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http://dx.doi.org/10.1002/chem.202000335DOI Listing
June 2020

DMT1 Inhibitors Kill Cancer Stem Cells by Blocking Lysosomal Iron Translocation.

Chemistry 2020 Jun 26;26(33):7369-7373. Epub 2020 May 26.

Institut Curie, 26 rue d'Ulm, 75248, Paris Cedex 05, France.

Cancer stem cells (CSC) constitute a cell subpopulation in solid tumors that is responsible for resistance to conventional chemotherapy, metastasis and cancer relapse. The natural product Salinomycin can selectively target this cell niche by directly interacting with lysosomal iron, taking advantage of upregulated iron homeostasis in CSC. Here, inhibitors of the divalent metal transporter 1 (DMT1) have been identified that selectively target CSC by blocking lysosomal iron translocation. This leads to lysosomal iron accumulation, production of reactive oxygen species and cell death with features of ferroptosis. DMT1 inhibitors selectively target CSC in primary cancer cells and circulating tumor cells, demonstrating the physiological relevance of this strategy. Taken together, this opens up opportunities to tackle unmet needs in anti-cancer therapy.
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http://dx.doi.org/10.1002/chem.202000159DOI Listing
June 2020

Whole-genome mapping of small-molecule targets for cancer medicine.

Curr Opin Chem Biol 2020 06 21;56:42-50. Epub 2020 Jan 21.

Institut Curie, 26 rue d'Ulm, 75248, Paris, Cedex 05, France; PSL Université Paris, France; Chemical Biology of Cancer Laboratory, CNRS UMR 3666, INSERM U1143, France. Electronic address:

Cancers display intratumoral and intertumoral heterogeneity, which poses challenges to small-molecule intervention. Studying drug responses on a whole-genome and transcriptome level using next-generation sequencing has revolutionized our understanding of how small molecules intervene in cells, which helps us to study and potentially predict treatment outcomes. Some small molecules act directly at the genomic level by targeting DNA or chromatin proteins. Here, we review recent advances in establishing whole-genome and transcriptome maps of small-molecule targets, comprising chromatin components or downstream events. We also describe recent advances in studying drug responses using single-cell RNA and DNA sequencing. Furthermore, we discuss how this fundamental research can be taken forward to devise innovative personalized treatment modalities.
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http://dx.doi.org/10.1016/j.cbpa.2019.12.005DOI Listing
June 2020

Image-Based Morphological Profiling Identifies a Lysosomotropic, Iron-Sequestering Autophagy Inhibitor.

Angew Chem Int Ed Engl 2020 03 24;59(14):5721-5729. Epub 2020 Jan 24.

Department of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany.

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.
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http://dx.doi.org/10.1002/anie.201913712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154763PMC
March 2020

Centromere Dysfunction Compromises Mitotic Spindle Pole Integrity.

Curr Biol 2019 09 5;29(18):3072-3080.e5. Epub 2019 Sep 5.

Institut Curie, PSL Research University, CNRS, UMR144, Biology of centrosomes and genetic instability lab, 75005 Paris, France. Electronic address:

Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2]. The CCAN is necessary for kinetochore assembly, a multiprotein complex that attaches spindle microtubules (MTs) and is required for chromosome segregation [3]. In most animal cells, the dominant site for MT nucleation in mitosis are the centrosomes, which are composed of two centrioles, surrounded by a protein-rich matrix of electron-dense pericentriolar material (PCM) [4]. The PCM is the site of MT nucleation during mitosis [5]. Even if centromeres and centrosomes are connected via MTs in mitosis, it is not known whether defects in either one of the two structures have an impact on the function of the other. Here, using high-resolution microscopy combined with rapid removal of CENP-A in human cells, we found that perturbation of centromere function impacts mitotic spindle pole integrity. This includes release of MT minus-ends from the centrosome, leading to PCM dispersion and centriole mis-positioning at the spindle poles. Mechanistically, we show that these defects result from abnormal spindle MT dynamics due to defective kinetochore-MT attachments. Importantly, restoring mitotic spindle pole integrity following centromere inactivation lead to a decrease in the frequency of chromosome mis-segregation. Overall, our work identifies an unexpected relationship between centromeres and maintenance of the mitotic pole integrity necessary to ensure mitotic accuracy and thus to maintain genetic stability.
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http://dx.doi.org/10.1016/j.cub.2019.07.052DOI Listing
September 2019

From controlling chemical bonding to deciphering and manipulating biological processes.

Bioorg Med Chem 2019 06 17;27(12):2281. Epub 2019 May 17.

CNRS, Institut Curie, 26 rue d'Ulm, 75005 Paris, France. Electronic address:

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http://dx.doi.org/10.1016/j.bmc.2019.05.027DOI Listing
June 2019

Diverse engineering.

Nat Chem 2019 06;11(6):499-500

Institut Curie, Paris, France.

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http://dx.doi.org/10.1038/s41557-019-0269-yDOI Listing
June 2019

Publisher Correction: PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

Nat Chem Biol 2019 05;15(5):549

Laboratoire de Biologie et Pharmacologie Appliquée, Ecole normale supérieure Paris-Saclay, Cachan, France.

In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.
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http://dx.doi.org/10.1038/s41589-019-0255-0DOI Listing
May 2019

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chembiochem 2019 04 25;20(7):968-973. Epub 2019 Feb 25.

PSL Université Paris, Institut Curie, CNRS UMR3666, INSERM U1143, 75005, Paris, France.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.
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http://dx.doi.org/10.1002/cbic.201900092DOI Listing
April 2019

PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

Nat Chem Biol 2019 04 11;15(4):358-366. Epub 2019 Feb 11.

Laboratoire de Biologie et Pharmacologie Appliquée, Ecole normale supérieure Paris-Saclay, Cachan, France.

Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.
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http://dx.doi.org/10.1038/s41589-019-0228-3DOI Listing
April 2019

Metformin reveals a mitochondrial copper addiction of mesenchymal cancer cells.

PLoS One 2018 6;13(11):e0206764. Epub 2018 Nov 6.

Chemical Biology of Cancer Team, Labellisée Ligue Contre le Cancer. PSL Research University, CNRS UMR3666 -INSERM U1143, Institut Curie, Paris, France.

The clinically approved drug metformin has been shown to selectively kill persister cancer cells through mechanisms that are not fully understood. To provide further mechanistic insights, we developed a drug surrogate that phenocopies metformin and can be labeled in situ by means of click chemistry. Firstly, we found this molecule to be more potent than metformin in several cancer cell models. Secondly, this technology enabled us to provide visual evidence of mitochondrial targeting with this class of drugs. A combination of fluorescence microscopy and cyclic voltammetry indicated that metformin targets mitochondrial copper, inducing the production of reactive oxygen species in this organelle, mitochondrial dysfunction and apoptosis. Importantly, this study revealed that mitochondrial copper is required for the maintenance of a mesenchymal state of human cancer cells, and that metformin can block the epithelial-to-mesenchymal transition, a biological process that normally accounts for the genesis of persister cancer cells, through direct copper targeting.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206764PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219783PMC
April 2019

PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers.

Cell Metab 2019 01 20;29(1):156-173.e10. Epub 2018 Sep 20.

Institut Curie, Stress and Cancer Laboratory, Equipe Labelisée par la Ligue Nationale contre le Cancer, PSL Research University, 26, rue d'Ulm, 75005 Paris, France; Inserm, U830, 26, rue d'Ulm, Paris 75005, France. Electronic address:

High-grade serous ovarian cancer (HGSOC) remains an unmet medical challenge. Here, we unravel an unanticipated metabolic heterogeneity in HGSOC. By combining proteomic, metabolomic, and bioergenetic analyses, we identify two molecular subgroups, low- and high-OXPHOS. While low-OXPHOS exhibit a glycolytic metabolism, high-OXPHOS HGSOCs rely on oxidative phosphorylation, supported by glutamine and fatty acid oxidation, and show chronic oxidative stress. We identify an important role for the PML-PGC-1α axis in the metabolic features of high-OXPHOS HGSOC. In high-OXPHOS tumors, chronic oxidative stress promotes aggregation of PML-nuclear bodies, resulting in activation of the transcriptional co-activator PGC-1α. Active PGC-1α increases synthesis of electron transport chain complexes, thereby promoting mitochondrial respiration. Importantly, high-OXPHOS HGSOCs exhibit increased response to conventional chemotherapies, in which increased oxidative stress, PML, and potentially ferroptosis play key functions. Collectively, our data establish a stress-mediated PML-PGC-1α-dependent mechanism that promotes OXPHOS metabolism and chemosensitivity in ovarian cancer.
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http://dx.doi.org/10.1016/j.cmet.2018.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331342PMC
January 2019

Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome.

Nat Commun 2018 04 27;9(1):1700. Epub 2018 Apr 27.

The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QN, UK.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, but devastating genetic disease characterized by segmental premature aging, with cardiovascular disease being the main cause of death. Cells from HGPS patients accumulate progerin, a permanently farnesylated, toxic form of Lamin A, disrupting the nuclear shape and chromatin organization, leading to DNA-damage accumulation and senescence. Therapeutic approaches targeting farnesylation or aiming to reduce progerin levels have provided only partial health improvements. Recently, we identified Remodelin, a small-molecule agent that leads to amelioration of HGPS cellular defects through inhibition of the enzyme N-acetyltransferase 10 (NAT10). Here, we show the preclinical data demonstrating that targeting NAT10 in vivo, either via chemical inhibition or genetic depletion, significantly enhances the healthspan in a Lmna HGPS mouse model. Collectively, the data provided here highlights NAT10 as a potential therapeutic target for HGPS.
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http://dx.doi.org/10.1038/s41467-018-03770-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5923383PMC
April 2018

Salinomycin kills cancer stem cells by sequestering iron in lysosomes.

Nat Chem 2017 10 16;9(10):1025-1033. Epub 2017 May 16.

Institut Curie, PSL Research University, Chemical Cell Biology Group, 26 rue d'Ulm, 75248 Paris Cedex 05, France.

Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.
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http://dx.doi.org/10.1038/nchem.2778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890907PMC
October 2017

Family-wide Analysis of the Inhibition of Arf Guanine Nucleotide Exchange Factors with Small Molecules: Evidence of Unique Inhibitory Profiles.

Biochemistry 2017 09 13;56(38):5125-5133. Epub 2017 Sep 13.

Laboratoire de Biologie et Pharmacologie Appliquée CNRS, Ecole Normale Supérieure Paris-Saclay , 61 avenue du président Wilson, 94235 Cachan, France.

Arf GTPases and their guanine nucleotide exchange factors (ArfGEFs) are major regulators of membrane traffic and organelle structure in cells. They are associated with a variety of diseases and are thus attractive therapeutic targets for inhibition by small molecules. Several inhibitors of unrelated chemical structures have been discovered, which have shown their potential in dissecting molecular pathways and blocking disease-related functions. However, their specificity across the ArfGEF family has remained elusive. Importantly, inhibitory responses in the context of membranes, which are critical determinants of Arf and ArfGEF cellular functions, have not been investigated. Here, we compare the efficiency and specificity of four structurally distinct ArfGEF inhibitors, Brefeldin A, SecinH3, M-COPA, and NAV-2729, toward six ArfGEFs (human ARNO, EFA6, BIG1, and BRAG2 and Legionella and Rickettsia RalF). Inhibition was assessed by fluorescence kinetics using pure proteins, and its modulation by membranes was determined with lipidated GTPases in the presence of liposomes. Our analysis shows that despite the intra-ArfGEF family resemblance, each inhibitor has a specific inhibitory profile. Notably, M-COPA is a potent pan-ArfGEF inhibitor, and NAV-2729 inhibits all GEFs, the strongest effects being against BRAG2 and Arf1. Furthermore, the presence of the membrane-binding domain in Legionella RalF reveals a strong inhibitory effect of BFA that is not measured on its GEF domain alone. This study demonstrates the value of family-wide assays with incorporation of membranes, and it should enable accurate dissection of Arf pathways by these inhibitors to best guide their use and development as therapeutic agents.
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http://dx.doi.org/10.1021/acs.biochem.7b00706DOI Listing
September 2017

Click chemistry enables preclinical evaluation of targeted epigenetic therapies.

Science 2017 06 15;356(6345):1397-1401. Epub 2017 Jun 15.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
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http://dx.doi.org/10.1126/science.aal2066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865750PMC
June 2017

An iron hand over cancer stem cells.

Autophagy 2017 Aug 14;13(8):1465-1466. Epub 2017 Jun 14.

b Institut Curie , PSL Research University, Chemical Cell Biology Group , Paris , France.

The paradigm of cancer stem cells (CSCs) defines the existence of cells exhibiting self-renewal and tumor-seeding capacity. These cells have been associated with tumor relapse and are typically resistant to conventional chemotherapeutic agents. Over the past decade, chemical biology studies have revealed a significant number of small molecules able to alter the proliferation of these cells in various settings. The natural product salinomycin has emerged as the most promising anti-CSC agent. However, an explicit mechanism of action has not yet been characterized, in particular due to the pleiotropic responses salinomycin is known for. In this punctum, we describe our recent discovery that salinomycin and the more potent synthetic derivative we named ironomycin sequester lysosomal iron. We found that these compounds, by blocking iron translocation, induce an iron-depletion response leading to a lysosomal degradation of ferritin followed by an iron-mediated lysosomal production of reactive oxygen species (ROS) and a cell death pathway that resembles ferroptosis. These unprecedented findings identified iron homeostasis and iron-mediated processes as potentially druggable in the context of CSCs.
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http://dx.doi.org/10.1080/15548627.2017.1327104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584845PMC
August 2017

Chromatin Regulates Genome Targeting with Cisplatin.

Angew Chem Int Ed Engl 2017 06 5;56(23):6483-6487. Epub 2017 May 5.

Institut Curie, PSL Research University, Chemical Cell Biology Group, 26 Rue d'Ulm, 75248, Paris Cedex 05, France.

Cisplatin derivatives can form various types of DNA lesions (DNA-Pt) and trigger pleiotropic DNA damage responses. Here, we report a strategy to visualize DNA-Pt with high resolution, taking advantage of a novel azide-containing derivative of cisplatin we named APPA, a cellular pre-extraction protocol and the labeling of DNA-Pt by means of click chemistry in cells. Our investigation revealed that pretreating cells with the histone deacetylase (HDAC) inhibitor SAHA led to detectable clusters of DNA-Pt that colocalized with the ubiquitin ligase RAD18 and the replication protein PCNA. Consistent with activation of translesion synthesis (TLS) under these conditions, SAHA and cisplatin cotreatment promoted focal accumulation of the low-fidelity polymerase Polη that also colocalized with PCNA. Remarkably, these cotreatments synergistically triggered mono-ubiquitination of PCNA and apoptosis in a RAD18-dependent manner. Our data provide evidence for a role of chromatin in regulating genome targeting with cisplatin derivatives and associated cellular responses.
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http://dx.doi.org/10.1002/anie.201701144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488169PMC
June 2017

Hepatocyte nuclear factor 1α suppresses steatosis-associated liver cancer by inhibiting PPARγ transcription.

J Clin Invest 2017 May 10;127(5):1873-1888. Epub 2017 Apr 10.

Worldwide epidemics of metabolic diseases, including liver steatosis, are associated with an increased frequency of malignancies, showing the highest positive correlation for liver cancer. The heterogeneity of liver cancer represents a clinical challenge. In liver, the transcription factor PPARγ promotes metabolic adaptations of lipogenesis and aerobic glycolysis under the control of Akt2 activity, but the role of PPARγ in liver tumorigenesis is unknown. Here we have combined preclinical mouse models of liver cancer and genetic studies of a human liver biopsy atlas with the aim of identifying putative therapeutic targets in the context of liver steatosis and cancer. We have revealed a protumoral interaction of Akt2 signaling with hepatocyte nuclear factor 1α (HNF1α) and PPARγ, transcription factors that are master regulators of hepatocyte and adipocyte differentiation, respectively. Akt2 phosphorylates and inhibits HNF1α, thus relieving the suppression of hepatic PPARγ expression and promoting tumorigenesis. Finally, we observed that pharmacological inhibition of PPARγ is therapeutically effective in a preclinical murine model of steatosis-associated liver cancer. Taken together, our studies in humans and mice reveal that Akt2 controls hepatic tumorigenesis through crosstalk between HNF1α and PPARγ.
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http://dx.doi.org/10.1172/JCI90327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409083PMC
May 2017

PSL Chemical Biology Symposia First 2016 Edition: When Chemistry and Biology Share the Language of Discovery.

Chembiochem 2017 05 30;18(10):883-887. Epub 2017 Mar 30.

Institut Curie, 26 rue d'Ulm, 75005, Paris, France.

Chemical biology, the science of understanding biological processes at the molecular level, has grown exponentially with the development of chemical strategies to manipulate and quantify biology with unprecedented precision. Recent advances presented at the Université Paris Sciences et Lettres symposium are discussed.
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http://dx.doi.org/10.1002/cbic.201700117DOI Listing
May 2017

Click Quantitative Mass Spectrometry Identifies PIWIL3 as a Mechanistic Target of RNA Interference Activator Enoxacin in Cancer Cells.

J Am Chem Soc 2017 02 23;139(4):1400-1403. Epub 2017 Jan 23.

Department of Molecular Biosciences, University of Texas at Austin , 2500 Speedway, Austin, Texas 78712, United States.

Enoxacin is a small molecule that stimulates RNA interference (RNAi) and acts as a growth inhibitor selectively in cancer but not in untransformed cells. Here, we used alkenox, a clickable enoxacin surrogate, coupled with quantitative mass spectrometry, to identify PIWIL3 as a mechanistic target of enoxacin. PIWIL3 is an Argonaute protein of the PIWI subfamily that is mainly expressed in the germline and that mediates RNAi through piRNAs. Our results suggest that cancer cells re-express PIWIL3 to repress RNAi through miRNAs and thus open a new opportunity for cancer-specific targeting.
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http://dx.doi.org/10.1021/jacs.6b11751DOI Listing
February 2017

Quinolizinium as a new fluorescent lysosomotropic probe.

Bioorg Med Chem Lett 2017 01 24;27(2):203-207. Epub 2016 Nov 24.

Institut Curie, PSL Research University, Organic Synthesis and Cell Biology Group, 26 rue d'Ulm, 75248 Paris Cedex 05, France; CNRS UMR3666, 75005 Paris, France; INSERM U1143, 75005 Paris, France.

We have synthesized a collection of quinolizinium fluorescent dyes for the purpose of cell imaging. Preliminary biological studies in human U2OS osteosarcoma cancer cells have shown that different functional groups appended to the cationic quinolizinium scaffold efficiently modulate photophysical properties but also cellular distribution. While quinolizinium probes are known nuclear staining reagents, we have identified a particular quinolizinium derivative salt that targets the lysosomal compartment. This finding raises the question of predictability of specific organelle targeting from structural features of small molecules.
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http://dx.doi.org/10.1016/j.bmcl.2016.11.074DOI Listing
January 2017

Synthesis of marmycin A and investigation into its cellular activity.

Nat Chem 2015 Sep 20;7(9):744-51. Epub 2015 Jul 20.

Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles du CNRS, 1 Avenue de la Terrasse, Gif sur-Yvette 91198, France.

Anthracyclines such as doxorubicin are used extensively in the treatment of cancers. Anthraquinone-related angucyclines also exhibit antiproliferative properties and have been proposed to operate via similar mechanisms, including direct genome targeting. Here, we report the chemical synthesis of marmycin A and the study of its cellular activity. The aromatic core was constructed by means of a one-pot multistep reaction comprising a regioselective Diels-Alder cycloaddition, and the complex sugar backbone was introduced through a copper-catalysed Ullmann cross-coupling, followed by a challenging Friedel-Crafts cyclization. Remarkably, fluorescence microscopy revealed that marmycin A does not target the nucleus but instead accumulates in lysosomes, thereby promoting cell death independently of genome targeting. Furthermore, a synthetic dimer of marmycin A and the lysosome-targeting agent artesunate exhibited a synergistic activity against the invasive MDA-MB-231 cancer cell line. These findings shed light on the elusive pathways through which anthraquinone derivatives act in cells, pointing towards unanticipated biological and therapeutic applications.
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http://dx.doi.org/10.1038/nchem.2302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892709PMC
September 2015

Defined Nutrient Diets Alter Susceptibility to Clostridium difficile Associated Disease in a Murine Model.

PLoS One 2015 16;10(7):e0131829. Epub 2015 Jul 16.

Division of Infectious Diseases and International Health, University of Virginia, Charlottesville, Virginia, United States of America.

Background: Clostridium difficile is a major identifiable and treatable cause of antibiotic-associated diarrhea. Poor nutritional status contributes to mortality through weakened host defenses against various pathogens. The primary goal of this study was to assess the contribution of a reduced protein diet to the outcomes of C. difficile infection in a murine model.

Methods: C57BL/6 mice were fed a traditional house chow or a defined diet with either 20% protein or 2% protein and infected with C. difficile strain VPI10463. Animals were monitored for disease severity, clostridial shedding and fecal toxin levels. Select intestinal microbiota were measured in stool and C. difficile growth and toxin production were quantified ex vivo in intestinal contents from untreated or antibiotic-treated mice fed with the different diets.

Results: C. difficile infected mice fed with defined diets, particularly (and unexpectedly) with protein deficient diet, had increased survival, decreased weight loss, and decreased overall disease severity. C. difficile shedding and toxin in the stool of the traditional diet group was increased compared with either defined diet 1 day post infection. Mice fed with traditional diet had an increased intestinal Firmicutes to Bacteroidetes ratio following antibiotic exposure compared with either a 2% or 20% protein defined nutrient diet. Ex vivo inoculation of cecal contents from antibiotic-treated mice showed decreased toxin production and C. difficile growth in both defined diets compared with a traditional diet.

Conclusions: Low protein diets, and defined nutrient diets in general, were found to be protective against CDI in mice. Associated diet-induced alterations in intestinal microbiota may influence colonization resistance and clostridial toxin production in a defined nutrient diet compared to a traditional diet, leading to increased survival. However, mechanisms which led to survival differences between 2% and 20% protein defined nutrient diets need to be further elucidated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504475PMC
April 2016
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