Publications by authors named "Fabio Gasparri"

33 Publications

EV20/NMS-P945, a Novel Thienoindole Based Antibody-Drug Conjugate Targeting HER-3 for Solid Tumors.

Pharmaceutics 2021 Apr 2;13(4). Epub 2021 Apr 2.

Department of Innovative Technologies in Medicine & Dentistry, University of Chieti-Pescara, 66100 Chieti, Italy.

HER-3 is becoming an attractive target for antibody-drug conjugate (ADC)-based therapy. Indeed, this receptor and its ligands are found to be overexpressed in several malignancies, and re-activation of its downstream signaling axis is known to play a critical role in modulating the sensitivity of targeted therapeutics in different tumors. In this study, we generated a novel ADC named EV20/NMS-P945 by coupling the anti-HER-3 antibody EV20 with a duocarmycin-like derivative, the thienoindole (TEI) NMS-P528, a DNA minor groove alkylating agent through a peptidic cleavable linker. This ADC showed target-dependent cytotoxic activity in vitro on several tumor cell lines and therapeutic activity in mouse xenograft tumor models, including those originating from pancreatic, prostatic, head and neck, gastric and ovarian cancer cells and melanoma. Pharmacokinetics and toxicological studies in monkeys demonstrated that this ADC possesses a favorable terminal half-life and stability and it is well tolerated. These data support further EV20/NMS-P945 clinical development as a therapeutic agent against HER-3-expressing malignancies.
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http://dx.doi.org/10.3390/pharmaceutics13040483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066800PMC
April 2021

The Coup-TFII orphan nuclear receptor is an activator of the γ-globin gene.

Haematologica 2021 Feb 1;106(2):474-482. Epub 2021 Feb 1.

Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, Milano, Italy.

The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of β-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and β039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the β/γ+β globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the β-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.
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http://dx.doi.org/10.3324/haematol.2019.241224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849756PMC
February 2021

Neutrophil Elastase Promotes Linker Cleavage and Paclitaxel Release from an Integrin-Targeted Conjugate.

Chemistry 2019 Feb 27;25(7):1696-1700. Epub 2018 Dec 27.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, I-20133, Milan, Italy.

This work takes advantage of one of the hallmarks of cancer, that is, the presence of tumor infiltrating cells of the immune system and leukocyte-secreted enzymes, to promote the activation of an anticancer drug at the tumor site. The peptidomimetic integrin ligand cyclo(DKP-RGD) was found to accumulate on the surface of α β integrin-expressing human renal cell carcinoma 786-O cells. The ligand was conjugated to the anticancer drug paclitaxel through a Asn-Pro-Val (NPV) tripeptide linker, which is a substrate of neutrophil-secreted elastase. In vitro linker cleavage assays and cell antiproliferative experiments demonstrate the efficacy of this tumor-targeting conjugate, opening the way to potential therapeutic applications.
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http://dx.doi.org/10.1002/chem.201805447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471013PMC
February 2019

Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury.

Mol Cancer Ther 2018 03 13;17(3):603-613. Epub 2017 Dec 13.

Oncology, Nerviano Medical Sciences, Nerviano, Milan, Italy.

Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRβ, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRβ inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative ICs correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0324DOI Listing
March 2018

Tumor Targeting with an isoDGR-Drug Conjugate.

Chemistry 2017 Jun 26;23(33):7910-7914. Epub 2017 May 26.

Dipartimento di Chimica, Università degli Studi di Milano, Via C. Golgi 19, 20133, Milano, Italy.

Herein we report the first example of an isoDGR-drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin α β . Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val-Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin α β receptor (IC =11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti-proliferative assays on two isogenic cancer cell lines characterized by different integrin α β expression: human glioblastoma U87 (α β +) and U87 β -KO (α β -). The isoDGR-PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD-PTX conjugate 4 (TI=2.4).
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http://dx.doi.org/10.1002/chem.201701844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488297PMC
June 2017

The development of high-content screening (HCS) technology and its importance to drug discovery.

Expert Opin Drug Discov 2016 28;11(5):501-14. Epub 2016 Mar 28.

a Department of Biology , Nerviano Medical Sciences S.r.l ., Nerviano , Milano , Italy.

Introduction: High-content screening (HCS) was introduced about twenty years ago as a promising analytical approach to facilitate some critical aspects of drug discovery. Its application has spread progressively within the pharmaceutical industry and academia to the point that it today represents a fundamental tool in supporting drug discovery and development.

Areas Covered: Here, the authors review some of significant progress in the HCS field in terms of biological models and assay readouts. They highlight the importance of high-content screening in drug discovery, as testified by its numerous applications in a variety of therapeutic areas: oncology, infective diseases, cardiovascular and neurodegenerative diseases. They also dissect the role of HCS technology in different phases of the drug discovery pipeline: target identification, primary compound screening, secondary assays, mechanism of action studies and in vitro toxicology.

Expert Opinion: Recent advances in cellular assay technologies, such as the introduction of three-dimensional (3D) cultures, induced pluripotent stem cells (iPSCs) and genome editing technologies (e.g., CRISPR/Cas9), have tremendously expanded the potential of high-content assays to contribute to the drug discovery process. Increasingly predictive cellular models and readouts, together with the development of more sophisticated and affordable HCS readers, will further consolidate the role of HCS technology in drug discovery.
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http://dx.doi.org/10.1517/17460441.2016.1165203DOI Listing
December 2016

A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds.

PLoS One 2015 28;10(10):e0141083. Epub 2015 Oct 28.

Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.

The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141083PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624791PMC
June 2016

Identification of thyroid tumor cell vulnerabilities through a siRNA-based functional screening.

Oncotarget 2015 Oct;6(33):34629-48

Molecular Mechanisms Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

The incidence of thyroid carcinoma is rapidly increasing. Although generally associated with good prognosis, a fraction of thyroid tumors are not cured by standard therapy and progress to aggressive forms for which no effective treatments are currently available. In order to identify novel therapeutic targets for thyroid carcinoma, we focused on the discovery of genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same degree for the viability of normal cells (non-oncogene addiction paradigm). We screened a siRNA oligonucleotide library targeting the human druggable genome in thyroid cancer BCPAP cell line in comparison with immortalized normal human thyrocytes (Nthy-ori 3-1). We identified a panel of hit genes whose silencing interferes with the growth of tumor cells, while sparing that of normal ones. Further analysis of three selected hit genes, namely Cyclin D1, MASTL and COPZ1, showed that they represent common vulnerabilities for thyroid tumor cells, as their inhibition reduced the viability of several thyroid tumor cell lines, regardless the histotype or oncogenic lesion. This work identified non-oncogenes essential for sustaining the phenotype of thyroid tumor cells, but not of normal cells, thus suggesting that they might represent promising targets for new therapeutic strategies.
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http://dx.doi.org/10.18632/oncotarget.5282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741478PMC
October 2015

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining.

Curr Protoc Cytom 2015 Apr 1;72:7.34.1-7.34.17. Epub 2015 Apr 1.

Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry.
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http://dx.doi.org/10.1002/0471142956.cy0734s72DOI Listing
April 2015

Synthesis and biological evaluation of RGD peptidomimetic-paclitaxel conjugates bearing lysosomally cleavable linkers.

Chemistry 2015 Apr 17;21(18):6921-9. Epub 2015 Mar 17.

Università degli Studi di Milano, Dipartimento di Chimica, Via C. Golgi, 19, 20133, Milan (Italy), Fax: (+39) 02-5031-4072.

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvβ3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVβ3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVβ3-) and its subclone CCRF-CEM αVβ3 (αVβ3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVβ3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.
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http://dx.doi.org/10.1002/chem.201500158DOI Listing
April 2015

Application of click chemistry conditions for 5-bromo-2'-deoxyuridine determination through Fenton and related reactions.

Curr Protoc Cytom 2015 Jan 5;71:7.43.1-7.43.17. Epub 2015 Jan 5.

Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.

Mixtures of ascorbate and copper used in certain click chemistry experimental conditions act as oxidizing agents, catalyzing the formation of reactive oxygen species through Fenton and related reactions. Hydroxyl radicals act as chemical nucleases, introducing DNA strand breaks that can be exploited for BrdU immunostaining in place of acid denaturation. This procedure is readily applicable to high content analysis and flow cytometry assays, and provides results comparable to click chemistry EdU cycloaddition and classical BrdU immunodetection. Importantly, this approach allows preservation of labile epitopes such as phosphoproteins. This unit describes an optimized method that successfully employs Fenton chemistry for simultaneous detection of phosphoproteins and BrdU in intact cells.
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http://dx.doi.org/10.1002/0471142956.cy0743s71DOI Listing
January 2015

Mcl-1 antagonism is a potential therapeutic strategy in a subset of solid cancers.

Exp Cell Res 2015 Mar 5;332(2):267-77. Epub 2014 Dec 5.

Nerviano Medical Sciences S.r.l. - Oncology, Viale Pasteur 10, I-20014 Nerviano, Milan, Italy.

Cancer cell survival is frequently dependent on the elevated levels of members of the Bcl-2 family of prosurvival proteins that bind to and inactivate BH3-domain pro-apoptotic cellular proteins. Small molecules that inhibit the protein-protein interactions between prosurvival and proapoptotic Bcl-2 family members (so-called "BH3 mimetics") have a potential therapeutic value, as indicated by clinical findings obtained with ABT-263 (navitoclax), a Bcl-2/Bcl-xL antagonist, and more recently with GDC-0199/ABT-199, a more selective antagonist of Bcl-2. Here, we report study results of the functional role of the prosurvival protein Mcl-1 against a panel of solid cancer cell lines representative of different tumor types. We observed silencing of Mcl-1 expression by small interfering RNAs (siRNAs) significantly reduced viability and induced apoptosis in almost 30% of cell lines tested, including lung and breast adenocarcinoma, as well as glioblastoma derived lines. Most importantly, we provide a mechanistic basis for this sensitivity by showing antagonism of Mcl-1 function with specific BH3 peptides against isolated mitochondria induces Bak oligomerization and cytochrome c release, therefore demonstrating that mitochondria from Mcl-1-sensitive cells depend on Mcl-1 for their integrity and that antagonizing Mcl-1 function is sufficient to induce apoptosis. Thus, our results lend further support for considering Mcl-1 as a therapeutic target in a number of solid cancers and support the rationale for development of small molecule BH3-mimetics antagonists of this protein.
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http://dx.doi.org/10.1016/j.yexcr.2014.11.022DOI Listing
March 2015

Discovery of 2-(cyclohexylmethylamino)pyrimidines as a new class of reversible valosine containing protein inhibitors.

J Med Chem 2014 Dec 12;57(24):10443-54. Epub 2014 Dec 12.

Oncology, Nerviano Medical Sciences S.r.l. , Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin-proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure-activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity.
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http://dx.doi.org/10.1021/jm501313xDOI Listing
December 2014

Optimization of diarylthiazole B-raf inhibitors: identification of a compound endowed with high oral antitumor activity, mitigated hERG inhibition, and low paradoxical effect.

ChemMedChem 2015 Feb 27;10(2):276-95. Epub 2014 Nov 27.

Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano (MI) (Italy).

Aberrant activation of the mitogen-activated protein kinase (MAPK)-mediated pathway components, RAF-MEK-ERK, is frequently observed in human cancers and clearly contributes to oncogenesis. As part of a project aimed at finding inhibitors of B-Raf, a key player in the MAPK cascade, we originally identified a thiazole derivative endowed with high potency and selectivity, optimal in vitro ADME properties, and good pharmacokinetic profiles in rodents, but that suffers from elevated hERG inhibitory activity. An optimization program was thus undertaken, focused mainly on the elaboration of the R(1) and R(2) groups of the scaffold. This effort ultimately led to N-(4-{2-(1-cyclopropylpiperidin-4-yl)-4-[3-(2,5-difluorobenzenesulfonylamino)-2-fluorophenyl]thiazol-5-yl}-pyridin-2-yl)acetamide (20), which maintains favorable in vitro and in vivo properties, but lacks hERG liability. Besides exhibiting potent antiproliferative activity against only cell lines bearing B-Raf V600E or V600D mutations, compound 20 also intriguingly shows a weaker "paradoxical" activation of MEK in non-mutant B-Raf cells than other known B-Raf inhibitors. It also demonstrates very good efficacy in vivo against the A375 xenograft melanoma model (tumor volume inhibition >90% at 10 mg kg(-1) ); it is therefore a suitable candidate for preclinical development.
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http://dx.doi.org/10.1002/cmdc.201402424DOI Listing
February 2015

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

Mol Oncol 2014 Dec 12;8(8):1495-507. Epub 2014 Jun 12.

Nerviano Medical Sciences S.r.l., Nerviano (Milan), Italy.

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.
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http://dx.doi.org/10.1016/j.molonc.2014.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528583PMC
December 2014

From "Click" to "Fenton" chemistry for 5-bromo-2'-deoxyuridine determination.

Cytometry A 2013 Nov 13;83(11):989-1000. Epub 2013 Aug 13.

Department of Biology Nerviano Medical Sciences, Via Pasteur 10, 20014, Nerviano, Italy.

Ascorbic acid (AA) and copper have been increasingly employed in flow cytometry (FCM) and high content analysis (HCA) since the introduction of "click chemistry" as a non-destructive alternative to classical 5-bromo-2'-deoxyuridine (BrdU) immunodetection for DNA synthesis and proliferation assays. Mixtures of ascorbate and catalytic copper, under certain experimental conditions, act as oxidizing agent, catalyzing the formation of reactive hydroxyl radicals through hydrogen peroxides decomposition via Fenton reaction. We developed a procedure for BrdU incorporation detection based on the use of AA and cupric ions as DNA damaging agents. Optimal DNA damaging conditions were identified and found to provide results comparable with "click" 5-ethynyl-deoxyuridine (EdU) cycloaddition approach and classical BrdU immunodetection. Scavenger agents were found to prevent hydroxyl-induced DNA damages, providing the proof-of-concept for the use of this procedure for DNA denaturation prior to BrdU detection. We demonstrated hydroxyl radicals' reaction to be readily applicable to HCA and FCM assays, for both classical BrdU immunostaining and EdU cycloaddition procedure. This technique was successfully employed for BrdU pulse-chase experiments and in multiparametric immunofluorescence assays for the simultaneous detection of labile phosphoproteins in intact cells. The use of AA/Cu prior to immunodetection for BrdU incorporation assays is a viable alternative to chemical/physical DNA denaturing agents (acids or heat), since it allows preservation of labile epitopes such as phosphoproteins, and over enzymatic agents (digestion with DNases) for its lower cost.
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http://dx.doi.org/10.1002/cyto.a.22343DOI Listing
November 2013

Highly multiplexed phenotypic imaging for cell proliferation studies.

J Biomol Screen 2014 Jan 29;19(1):145-57. Epub 2013 Jul 29.

1Cell Biology Department, Oncology Business Unit, Nerviano Medical Sciences S.r.l., Nerviano, Italy.

The application of multiplexed imaging technologies in phenotypic drug discovery (PDD) enables profiling of complex cellular perturbations in response to drug treatment. High-content analysis (HCA) is among the most pursued approaches in PDD, with a proven capability to identify compounds with a given cellular mechanism of action (MOA), as well as to unveil unexpected drug cellular activities. The ability of fluorescent image-based cytometric techniques to dissect the phenotypic heterogeneity of cell populations depends on the degree of multiplexing achievable. At present, most high-content assays employ up to four cellular markers separately detected in distinct fluorescence channels. We explored the possibility to increase HCA multiplexing through analysis of multiple proliferation markers in the same fluorescence channel by taking advantage of the different timing of antigen appearance during the cell cycle, or differential intracellular localization. Simultaneous analysis of DAPI staining and five immunofluorescence markers (BrdU incorporation, active caspase-3, phospho-histone H3, phospho-S6, and Ki-67) resulted in the first six-marker high-content assay readily applicable to compound MOA studies. This approach allows detection of rare cell subpopulations, unveiling a high degree of phenotypic heterogeneity in exponentially growing cell cultures and variability in the individual cell response to antiproliferative drugs.
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http://dx.doi.org/10.1177/1087057113495712DOI Listing
January 2014

Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death.

Nat Chem Biol 2013 Sep 28;9(9):548-56. Epub 2013 Jul 28.

Business Unit Oncology, Nerviano Medical Sciences, Nerviano, Italy.

VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.
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http://dx.doi.org/10.1038/nchembio.1313DOI Listing
September 2013

Cell line identity finding by fingerprinting, an optimized resource for short tandem repeat profile authentication.

Genet Test Mol Biomarkers 2013 Mar 28;17(3):254-9. Epub 2013 Jan 28.

Business Unit Oncology, Nerviano Medical Sciences S.r.l., Nerviano (MI), Italy.

The generation of biological data on wide panels of tumor cell lines is recognized as a valid contribution to the cancer research community. However, research laboratories can benefit from this knowledge only after the identity of each individual cell line used in the experiments is verified and matched to external sources. Among the methods employed to assess cell line identity, DNA fingerprinting by profiling Short Tandem Repeat (STR) at variable loci has become the method of choice. However, the analysis of cancer cell lines is sometimes complicated by their intrinsic genetic instability, resulting in multiple allele calls per locus. In addition, comparison of data across different sources must deal with the heterogeneity of published profiles both in terms of number and type of loci used. The aim of this work is to provide the scientific community a homogeneous reference dataset for 300 widely used tumor cell lines, profiled in parallel on 16 loci. This large dataset is interfaced with an in-house developed software tool for Cell Line Identity Finding by Fingerprinting (CLIFF), featuring an original identity score calculation, which facilitates the comparison of STR profiles from different sources and enables accurate calls when multiple loci are present. CLIFF additionally allows import and query of proprietary STR profile datasets.
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http://dx.doi.org/10.1089/gtmb.2012.0359DOI Listing
March 2013

Cell-based assays--Informa Life Sciences' Fifth Annual Conference--Label-free cell-based assays and high-content analysis in drug discovery.

Authors:
Fabio Gasparri

IDrugs 2010 Aug;13(8):523-6

Nerviano Medical Sciences Srl, Milan, Italy.

Informa Life Sciences' Fifth Annual Conference on Cell-Based Assays, held in Cologne, Germany, included topics covering new technical developments in the field of cell-based assays. This conference report highlights selected presentations on label-free cell-based assays and the application of high-content analysis to drug discovery.
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August 2010

Cell-based assays--Informa Life Sciences' Fifth Annual Conference--Cell-based assays for compound screening and 3D assays.

Authors:
Fabio Gasparri

IDrugs 2010 Aug;13(8):520-2

Nerviano Medical Sciences Srl, Milan, Italy.

Informa Life Sciences' Fifth Annual Conference on Cell-Based Assays, held in Cologne, Germany, included topics covering new technical developments in the field of cell-based assays. This conference report highlights selected presentations on cell-based assays for compound screening and 3D cell-based assays.
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August 2010

Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan assays.

Eur J Cell Biol 2009 Dec 2;88(12):779-85. Epub 2009 Sep 2.

Biology Department, Oncology, Nerviano Medical Sciences, Nerviano, Italy.

Thymidine kinase 1 (TK1) is a DNA salvage enzyme involved in the synthesis of thymidine triphosphate needed during S phase. Although TK1 has been utilized as a cell proliferation marker for many years no well-characterized antibodies are available. The preparation and properties of two types of poly- and monoclonal anti-TK1 peptide antibodies are described and they are used to determine the levels of TK1 in intact cells. Expression of TK1, c-fos, cyclin B1, Ki67, phosphorylated histone H3, phosphorylated ribosomal protein S6, as well as bromodeoxyuridine (BrdU) incorporation in human normal dermal fibroblast cultures were studied with high-content ArrayScan fluorescence microscopy. The levels of TK1 increased 6-7h after serum re-addition to starved cells as they passed through G1, S and G2/M phases, which was earlier than the increase in Ki67 protein levels and before BrdU incorporation was detected. Thus, a population of activated G1 cells with high TK1 and low Ki67 expression could be identified and their role in cell proliferation can now be clarified.
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http://dx.doi.org/10.1016/j.ejcb.2009.06.005DOI Listing
December 2009

An overview of cell phenotypes in HCS: limitations and advantages.

Authors:
Fabio Gasparri

Expert Opin Drug Discov 2009 Jun;4(6):643-57

Department of Cell Biology, Oncology Business Unit, Nerviano Medical Sciences S.r.l., Viale Pasteur 10, 20014, Nerviano (Milano), Italy +39 0331 581706 ; +39 0331 581233 ;

Background: High-content screening (HCS) defines a series of cell-based multiparametric approaches for analysis at the single-cell level. In recent years, HCS has been increasingly pursued in the drug discovery field, adding to the repertoire of assay type, or increasing throughput in applications such as compound screening and mechanism of action studies, as well as for target identification/validation (siRNA screening). Obviously, as cells represent the objects of high-content assays, the outcome of any HCS assay is determined by the cell type: the choice of the most suitable cellular model for a given assay is a critical step that must follow biological and technical criteria.

Method: Here, I discuss these criteria and report a systematic survey of cell types used so far in HCS, with particular emphasis on their strengths and drawbacks. I also illustrate my expectations for future advances on cellular models used in HCS.

Conclusion: Despite the plethora of cell types potentially suitable for HCS, so far only a handful of cellular models (particularly human cancer cell lines) account for the great majority of HCS assays. In the future, the introduction of novel cell types, including engineered and primary cells, will further expand the potential of HCS for systems biology and drug discovery.
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http://dx.doi.org/10.1517/17460440902992870DOI Listing
June 2009

Cell proliferation method: click chemistry based on BrdU coupling for multiplex antibody staining.

Curr Protoc Cytom 2008 Jul;Chapter 7:Unit7.34

Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle (see UNIT 7.7). However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by an alkenyl deoxyuridine (5-ethynyl-2'-deoxyuridine, EdU), which is incorporated into DNA. The nucleotide exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is preferred over traditional BrdU detection since it is more sensitive and allows multicolor and multiplex analysis in FCM and imaging.
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http://dx.doi.org/10.1002/0471142956.cy0734s45DOI Listing
July 2008

High-content analysis of kinase activity in cells.

Comb Chem High Throughput Screen 2008 Aug;11(7):523-36

Department of Cell Biology, Oncology Business Unit, Nerviano Medical Sciences Srl, Nerviano, Milano, Italy.

High-content analysis (HCA) is a term used to describe techniques involving multiplexed analysis of fluorescent markers to measure multiple cellular responses to biological stimuli or drug treatment. HCA is usually based on automated microscopy or related technologies, and its value lies in providing multiparametric information on single cells within a population. During the last decade, several HCA approaches have been developed and applied to assess cellular mechanism of action of pharmacologically relevant compounds identified through biochemical screening or similar in vitro methods. With automation and instrument development, these approaches have evolved to the extent that the technique is now routinely used in screening applications, including primary HTS on compound collections. Here, we review the field and discuss in particular the application of HCA to the discovery of small molecule inhibitors targeting kinases which are implicated in Oncology.
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http://dx.doi.org/10.2174/138620708785204126DOI Listing
August 2008

A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining.

Cytometry A 2008 Jul;73(7):626-36

Department of Cell Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Nerviano (Mi), Italy.

Quantification of BrdU incorporation into DNA is a widely used technique to assess the cell cycle status of cells. DNA denaturation is required for BrdU detection with the drawback that most protein epitopes are destroyed and classical antibody staining techniques for multiplex analysis are not possible. To address this issue we have developed a novel method that overcomes the DNA denaturation step but still allows detection of BrdU. Cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine, which is incorporated into DNA. The exposed nucleotide alkyne group of DNA was then derivatized in physiologic conditions by the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) using BrdU azides. The resulting DNA-bound bromouracil moiety was subsequently detected by commercial anti-BrdU mAb without the need for a denaturation step. Continuous labeling with EdU showed a slightly increased anti-proliferative activity compared to BrdU. However, using a lower concentration of EdU for labeling can compensate for this. Alkynyl tags could be detected quickly by a highly specific reaction using BrdU azides. Fluorescence quenching by the DNA dye PI using both BrdU azides was negligible. Our labeling method is suitable for FCM and HCA and shows a higher signal to noise ratio than other methods. This method also allowed multiplex analysis by simultaneous detection of EdU-BrdU, caspase-3, and phospho-histone 3 mAbs, proving sensitivity and feasibility of this new technique. In addition, it has the potential for use in vivo, as exemplified for bone marrow studies. We have established a new method to determine the position of cells in the cell cycle. This is superior when compared to traditional BrdU detection since it allows multiplex analysis, is more sensitive and shows less quenching with PI. The method provides new opportunities to investigate changes in protein expression at different cell cycle stages using pulse labeling experiments.
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http://dx.doi.org/10.1002/cyto.a.20582DOI Listing
July 2008

Bortezomib inhibits nuclear factor-kappaB dependent survival and has potent in vivo activity in mesothelioma.

Clin Cancer Res 2007 Oct;13(19):5942-51

Institute of Internal Medicine and Medical Oncology, IRCCS Policlinico San Matteo University Hospital, 1-27100 Pavia [corrected] Italy.

Purpose: Purpose of this study has been the assessment of nuclear factor-kappaB (NF-kappaB) as a survival factor in human mesothelial cells (HMC), transformed HMC and malignant mesothelioma (MMe) cells. We aimed at verifying whether the proteasome inhibitor Bortezomib could abrogate NF-kappaB activity in MMe cells, leading to tumor cell death and may be established as a novel treatment for this aggressive neoplasm.

Experimental Design: In HMC and MMe cells, NF-kappaB nuclear translocation and DNA binding were studied by electrophoretic mobility shift assay, following treatment with tumor necrosis factor-alpha (TNF-alpha). The IKK inhibitor Bay11-7082 was also tested to evaluate its effects on HMC, transformed HMC, and MMe cell viability upon exposure to asbestos fibers. Following Bortezomib treatment, cytotoxicity of MMe cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, whereas apoptosis and cell-cycle blockade were investigated by high-content analysis. Bortezomib was also given to mice bearing i.p. xenografts of MMe cells, and its effects on tumor growth were evaluated.

Results: Here, we show that NF-kappaB activity is a constitutive survival factor in transformed HMC, MMe cells, and acts as a survival factor in HMC exposed to asbestos fibers. Bortezomib inhibits NF-kappaB activity in MMe cells and induces cell cycle blockade and apoptosis in vitro as well as tumor growth inhibition in vivo.

Conclusions: Inhibition of NF-kappaB constitutive activation in MMe cells by Bortezomib resulted in in vitro cytotoxicity along with apoptosis and in vivo tumor regression. Our results support the use of Bortezomib in the treatment of MMe and has led to a phase II clinical trial currently enrolling in Europe.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-0536DOI Listing
October 2007

Cell-cycle inhibitor profiling by high-content analysis.

Adv Exp Med Biol 2007 ;604:137-48

Department of Biology, Nerviano Medical Sciences, Milano, Italy.

The discovery of agents which disrupt cancer cell division by specifically targeting key components of the cell-cycle machinery represents a major focus of recent drug discovery efforts in Oncology. The drug discovery process can be greatly enhanced by multiparametric cellular analysis which can assist in confirmation, often in a few multiplexed assays, of the mechanism of action (MOA) of compounds identified through biochemical screening or similar in vitro methods. High-Content Analysis (HCA) is a technique based on automated microscopy which enables multiparametric analysis of fluorescent indicators to define cellular responses to compound treatment. Several distinct fluorescence channels can be acquired and analyzed within a single measure in the same cell population. Here we present a multiparametric HCA approach to characterize potential cell-cycle inhibitors in osteosarcoma U-2 OS adherent cell cultures. This approach allows monitoring of compound-induced cell-cycle perturbations by analyzing specific cellular markers such as nuclear morphology, DNA content or histone H3 phosphorylation. Moreover, the induction of DNA damage response or apoptosis can also be readily evaluated. By considering the profile of the investigated cellular markers at different compound concentrations, a fingerprint defines the cellular and molecular phenotype associated with each compound.
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http://dx.doi.org/10.1007/978-0-387-69116-9_13DOI Listing
September 2007

Multiparametric cell cycle analysis by automated microscopy.

J Biomol Screen 2006 Sep 14;11(6):586-98. Epub 2006 Jul 14.

Biology Department, Oncology, Nerviano Medical Sciences, Nerviano, Italy.

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.
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http://dx.doi.org/10.1177/1087057106289406DOI Listing
September 2006

Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader.

Drug Discov Today 2005 ;Suppl:31-42

DRO-Oncology, Pharmacology Department, Pharmacia Corporation, Nerviano, Italy.

High-throughput cell-based assays are becoming a powerful approach in the drug discovery process. The ArrayScan high-content screening (HCS) reader is a cytometer based on a fully automated fluorescence microscope that is able to obtain quantitative information on the intensity and localization of fluorescence signals within single cells over a wide cell population. The aim of this work was to set up an automated HCS multiparameter analysis for the quantification of the in vitro proliferation index of normal human dermal fibroblast (NHDF) cultures. The authors stimulated starved NHDF with insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, or serum, and they quantified the proliferation index by measuring the expression of Ki-67 antigen, the incorporation of bromodeoxyuridine (BrdU), and the phosphorylation of the retinoblastoma protein (pRb). This approach also allowed quantification of the mitotic index by phospho-histone H3 staining and the percentage of cells in the S-phase by BrdU incorporation. The proliferation data from the ArrayScan assays were validated by comparison with a reference enzyme-linked immunosorbent assay (ELISA) and by flow cytometry. The measured proliferation indices were highly reproducible in repeated measures and independent experiments. The authors therefore propose that the ArrayScan HCS system could be used for high-throughput multiparameter analysis and quantification of the proliferation of cellular cultures.
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June 2013