Publications by authors named "Christophe Antczak"

40 Publications

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

PLoS One 2020 24;15(8):e0235551. Epub 2020 Aug 24.

Novartis Institutes for Biomedical Research, Basel, Switzerland.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0235551PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446895PMC
September 2020

Fluopack screening platform for unbiased cellular phenotype profiling.

Sci Rep 2020 02 7;10(1):2097. Epub 2020 Feb 7.

Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

Gene and compound functions are often interrogated by perturbation. However, we have limited methods to capture associated phenotypes in an unbiased and holistic manner. Here, we describe Fluopack screening as a novel platform enabling the profiling of subcellular phenotypes associated with perturbation. Our approach leverages imaging of a panel of fluorescent chemical probes to survey cellular processes in an unbiased and high throughput fashion. Segmentation-free, whole image analysis applied to Fluopack images identifies probes revealing distinct phenotypes upon perturbation, thereby informing on the function and mechanism of action of perturbagens. This chemical biology approach allows to interrogate phenotypes that tend to be overlooked by other methods, such as lipid trafficking and ion concentration inside the cell. Fluopack screening is a powerful approach to study orphan protein function, as exemplified by the characterization of TMEM41B as novel regulator of lipid mobilization.
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http://dx.doi.org/10.1038/s41598-020-58861-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005823PMC
February 2020

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

Nat Chem Biol 2019 02 14;15(2):179-188. Epub 2019 Jan 14.

Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
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http://dx.doi.org/10.1038/s41589-018-0200-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251565PMC
February 2019

TMEM41B is a novel regulator of autophagy and lipid mobilization.

EMBO Rep 2018 09 20;19(9). Epub 2018 Aug 20.

Novartis Institutes for BioMedical Research, Basel, Switzerland

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.
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http://dx.doi.org/10.15252/embr.201845889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123663PMC
September 2018

How Phenotypic Screening Influenced Drug Discovery: Lessons from Five Years of Practice.

Assay Drug Dev Technol 2017 Aug/Sep;15(6):239-246. Epub 2017 Aug 11.

1 Novartis Institutes for BioMedical Research (NIBR) , Chemical Biology and Therapeutics (CBT), Basel, Switzerland .

Since 2011, phenotypic screening has been a trend in the pharmaceutical industry as well as in academia. This renaissance was triggered by analyses that suggested that phenotypic screening is a superior strategy to discover first-in-class drugs. Despite these promises and considerable investments, pharmaceutical research organizations have encountered considerable challenges with the approach. Few success stories have emerged in the past 5 years and companies are questioning their investment in this area. In this contribution, we outline what we have learned about success factors and challenges of phenotypic screening. We then describe how our efforts in phenotypic screening have influenced our approach to drug discovery in general. We predict that concepts from phenotypic screening will be incorporated into target-based approaches and will thus remain influential beyond the current trend.
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http://dx.doi.org/10.1089/adt.2017.796DOI Listing
May 2018

Evaluation of Compound Optical Interference in High-Content Screening.

SLAS Discov 2018 04 3;23(4):321-329. Epub 2017 May 3.

2 HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Compound optical interference remains an inherent problem in chemical screening and has been well documented for biochemical assays and less so for automated microscopy-based assays. It has also been the assumption that the latter should not suffer from such interference because of the washing steps involved in the process, thus eliminating the residual nonspecific compound effects. Instead, these compounds may have no relevance to the actual target, and as such, compound optical interference contributes to a number of false-positives, resulting in a high attrition rate during subsequent follow-up studies. In this report, we analyze the outcome of a high-content screen using enhanced green fluorescent protein as a reporter in a gain-of-function cell-based assay in search of modulators of the micro RNA (miRNA) biogenesis pathway. Using a previously validated image-based biosensor, we screened a diverse library collection of ~315,000 compounds covering natural and synthetic derivatives in which 1130 positives were identified to enhance green fluorescence expression. Lateral confirmation and dose-response studies revealed that all of these compounds were the result of optical interference and not specific inhibition of miRNA biogenesis. Here, we highlight the chemical classes that are susceptible to compound optical interference and discuss their implications in automated microscopy-based assays.
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http://dx.doi.org/10.1177/2472555217707725DOI Listing
April 2018

ABCG2 regulates self-renewal and stem cell marker expression but not tumorigenicity or radiation resistance of glioma cells.

Sci Rep 2016 07 26;6:25956. Epub 2016 Jul 26.

Human Biology Division, Solid Tumor and Translational Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Glioma cells with stem cell traits are thought to be responsible for tumor maintenance and therapeutic failure. Such cells can be enriched based on their inherent drug efflux capability mediated by the ABC transporter ABCG2 using the side population assay, and their characteristics include increased self-renewal, high stem cell marker expression and high tumorigenic capacity in vivo. Here, we show that ABCG2 can actively drive expression of stem cell markers and self-renewal in glioma cells. Stem cell markers and self-renewal was enriched in cells with high ABCG2 activity, and could be specifically inhibited by pharmacological and genetic ABCG2 inhibition. Importantly, despite regulating these key characteristics of stem-like tumor cells, ABCG2 activity did not affect radiation resistance or tumorigenicity in vivo. ABCG2 effects were Notch-independent and mediated by diverse mechanisms including the transcription factor Mef. Our data demonstrate that characteristics of tumor stem cells are separable, and highlight ABCG2 as a potential driver of glioma stemness.
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http://dx.doi.org/10.1038/srep25956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960591PMC
July 2016

Proteasome Addiction Defined in Ewing Sarcoma Is Effectively Targeted by a Novel Class of 19S Proteasome Inhibitors.

Cancer Res 2016 08 2;76(15):4525-34. Epub 2016 Jun 2.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York.

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525-34. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-1040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5484002PMC
August 2016

Azaphilones from an Acid Mine Extremophile Strain of a Pleurostomophora sp.

J Nat Prod 2015 Dec 7;78(12):2917-23. Epub 2015 Dec 7.

HTS Core Facility, Memorial Sloan-Kettering Cancer Center , New York, New York 10065, United States.

An extremophilic fungus identified as a Pleurostomophora sp. was isolated from the Berkeley Pit, an acid mine waste lake. When grown in liquid culture, the fungus produced berkchaetoazaphilones A-C (1, 2, and 5), the red pigment berkchaetorubramine (6), and the known compound 4-(hydroxymethyl)quinoline. These compounds were evaluated as inhibitors of matrix metalloproteinase-3, caspase-1, and proinflammatory cytokine production in induced THP-1 cells. Berkchaetoazaphilone B (2) inhibited IL-1β, TNFα, and IL-6 production in the induced inflammasome assay and was cytotoxic toward human retinoblastoma cell line Y79 (IC50 = 1.1 μM), leukemia cell lines CCRF-CEM and SR, and the melanoma cell line LOX IMVI (IC50 = 10 μM).
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http://dx.doi.org/10.1021/acs.jnatprod.5b00519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156323PMC
December 2015

A High-Content Assay to Screen for Modulators of EGFR Function.

Methods Mol Biol 2016 ;1360:97-106

HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Cell-based assays have the potential and advantage to identify cell-permeable modulators of kinase function, and hence provide an alternative to the conventional enzymatic activity-driven discovery approaches that rely on purified recombinant kinase catalytic domains. Here, we describe a domain-based high-content biosensor approach to study endogenous EGFR activity whereby EGF-induced receptor activation, subsequent trafficking, and internalization are imaged and quantified using time-dependent granule formation in cells. This method can readily be used to search for EGFR modulators in both chemical and RNAi screening; with potential applicability to other receptor tyrosine kinases.
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http://dx.doi.org/10.1007/978-1-4939-3073-9_8DOI Listing
August 2016

Challenges and opportunities toward enabling phenotypic screening of complex and 3D cell models.

Future Med Chem 2015 ;7(4):513-25

Advanced Assays, Genomics Institute of the Novartis Research Foundation (GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.

Increasingly, organotypic cellular platforms are being recognized as useful tools in drug discovery. This review offers an industry-centric perspective on the benefits of emerging complex cell models over conventional 2D systems, as well as the challenges and opportunities for incorporating these multidimensional platforms into high-density formats. We particularly highlight the need for novel chemical sensors to noninvasively quantitate 3D structures in real time, and we contend that the use of more focused chemical and genomics libraries will enable screening of complex cell models derived from primary and induced pluripotent stem cells. Finally, we offer outlooks on several emerging technologies that show great potential for future integration of complex cell systems into contemporary drug screening.
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http://dx.doi.org/10.4155/fmc.14.163DOI Listing
January 2016

A 1536-well fluorescence polarization assay to screen for modulators of the MUSASHI family of RNA-binding proteins.

Comb Chem High Throughput Screen 2014 ;17(7):596-609

(Michael G. Kharas) Molecular Pharmacology & Chemistry Program, MSKCC, New York, USA.

RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10 μM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135234PMC
http://dx.doi.org/10.2174/1386207317666140609122714DOI Listing
April 2015

Chemical & RNAi screening at MSKCC: a collaborative platform to discover & repurpose drugs to fight disease.

Comb Chem High Throughput Screen 2014 May;17(4):298-318

HTS Core Facility, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

Memorial Sloan Kettering Cancer Center (MSKCC) has implemented the creation of a full service state-of-the-art High-throughput Screening Core Facility (HTSCF) equipped with modern robotics and custom-built screening data management resources to rapidly store and query chemical and RNAi screening data outputs. The mission of the facility is to provide oncology clinicians and researchers alike with access to cost-effective HTS solutions for both chemical and RNAi screening, with an ultimate goal of novel target identification and drug discovery. HTSCF was established in 2003 to support the institution's commitment to growth in molecular pharmacology and in the realm of therapeutic agents to fight chronic diseases such as cancer. This endeavor required broad range of expertise in technology development to establish robust and innovative assays, large collections of diverse chemical and RNAi duplexes to probe specific cellular events, sophisticated compound and data handling capabilities, and a profound knowledge in assay development, hit validation, and characterization. Our goal has been to strive for constant innovation, and we strongly believe in shifting the paradigm from traditional drug discovery towards translational research now, making allowance for unmet clinical needs in patients. Our efforts towards repurposing FDA-approved drugs fructified when digoxin, identified through primary HTS, was administered in the clinic for treatment of stage Vb retinoblastoma. In summary, the overall aim of our facility is to identify novel chemical probes, to study cellular processes relevant to investigator's research interest in chemical biology and functional genomics, and to be instrumental in accelerating the process of drug discovery in academia.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050342PMC
http://dx.doi.org/10.2174/1386207317666140323132222DOI Listing
May 2014

Phomopsolides and related compounds from the alga-associated fungus, Penicillium clavigerum.

Nat Prod Commun 2014 Jan;9(1):87-90

For the past fifteen years we have studied the secondary metabolites of extremophilic fungi from the Berkeley Pit, an abandoned acid mine waste lake. Fungi associated with an acid-tolerant alga have also been harvested from the Pit. Penicillium clavigerum Demelius was isolated from the green alga Chlorella vulgaris Beyerinck [Beijerinck]. In culture it produced the known compounds phomfuranone (1), patulin (2), dimethylphthalides (3) and (4), phomopsolide A (5), phomopsolide C (6), phomopsolide B (7), phomopsolide E (8), phomopsolide F (9), and phompyrone (10) and the new compound berkbenzofuran thioester (11). Compounds 5 and 6 were potent inhibitors (IC50 < 10 microM) of specific and established human cancer cell lines.
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January 2014

A high-content assay strategy for the identification and profiling of ABCG2 modulators in live cells.

Assay Drug Dev Technol 2014 Jan-Feb;12(1):28-42. Epub 2013 Aug 30.

1 High-Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center , New York, New York.

ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which has been implicated in resistance to various chemotherapeutic agents. Though a number of cell-based assays to screen for inhibitors have been reported, they do not provide a content-rich platform to discriminate toxic and autofluorescent compounds. To fill this gap, we developed a live high-content cell-based assay to identify inhibitors of ABCG2-mediated transport and, at the same time, assess their cytotoxic effect and potential optical interference. We used a pair of isogenic U87MG human glioblastoma cell lines, with one stably overexpressing the ABCG2 transporter. JC-1 (J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol carbocyanine iodide) was selected as the optimal reporter substrate for ABCG2 activity, and the resulting assay was characterized by a Z' value of 0.50 and a signal-to-noise (S/N) ratio of 14 in a pilot screen of ∼ 7,000 diverse chemicals. The screen led to the identification of 64 unique nontoxic positives, yielding an initial hit rate of 1%, with 58 of them being confirmed activity. In addition, treatment with two selected confirmed positives suppressed the side population of U87MG-ABCG2 cells that was able to efflux the Hoechst dye as measured by flow cytometry, confirming that they constitute potent new ABCG2 transporter inhibitors. Our results demonstrate that our live cell and content-rich platform enables the rapid identification and profiling of ABCG2 modulators, and this new strategy opens the door to the discovery of compounds targeting the expression and/or trafficking of ABC transporters as an alternative to functional inhibitors that failed in the clinic.
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http://dx.doi.org/10.1089/adt.2013.521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934439PMC
October 2014

A high content assay to assess cellular fitness.

Comb Chem High Throughput Screen 2014 Jan;17(1):12-24

HTS Core Facility, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

A universal process in experimental biology is the use of engineered cells; more often, stably or transiently transfected cells are generated for the purpose. Therefore, it is important that cell health assessment is conducted to check for stress mediated by induction of heat shock proteins (Hsps). For this purpose, we have developed an integrated platform that would enable a direct assessment of transfection efficiency (TE) combined with cellular toxicity and stress response. We make use of automated microscopy and high content analysis to extract from the same well a multiplexed readout to assess and determine optimal chemical transfection conditions. As a proof of concept, we investigated seven commercial reagents, in a matrix of dose and time, to study transfection of an EGFP DNA plasmid into HeLa cells and their consequences on health and fitness; where we scored for cellular proliferation, EGFP positive cells, and induction of Hsp10 and Hsp70 as makers of stress responses. FuGENE HD emerged as the most optimal reagent with no apparent side effects suitable for performing microtiter based miniaturized transfection for both chemical and RNAi screening. In summary, we report on a high content assay method to assess cellular overall fitness upon chemical transfection.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947212PMC
http://dx.doi.org/10.2174/13862073113169990056DOI Listing
January 2014

A synergetic screening approach with companion effector for combination therapy: application to retinoblastoma.

PLoS One 2013 19;8(3):e59156. Epub 2013 Mar 19.

HTS Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, New York, United States of America.

For many cancers, the lack of potency and the toxicity of current drugs limits the dose achievable in patients and the efficacy of treatment. Among them, retinoblastoma is a rare cancer of the eye for which better chemotherapeutic options are needed. Combination therapy is a compelling approach to enhance the efficacy of current treatment, however clinical trials to test rationally designed combinations of approved drugs are slow and expensive, and limited by our lack of in-depth knowledge of drug specificity. Since many patients already turn to nutraceuticals in hopes of improving their condition, we hypothesized that certain approved drugs could potentially synergize with widely consumed supplements. Following this hypothesis, we devised an alternative screening strategy aimed at taking advantage of a bait compound such as a nutraceutical with potential therapeutic benefits but low potency, by screening chemical libraries for approved drugs that synergize with this companion effector. As a proof of concept, we sought to identify approved drugs with synergetic therapeutic effects toward retinoblastoma cells in combination with the antioxidant resveratrol, popular as a supplement. We systematically tested FDA-approved drugs and known bioactives seeking to identify such pairs, which led to uncovering only a few additive combinations; but to our surprise, we identified a class of anticancer drugs widely used in the clinic whose therapeutic effect is antagonized with resveratrol. Our observations could explain in part why some patients do not respond well to treatment. Our results validate this alternative approach, and we expect that our companion effector strategy could significantly impact both drug discovery and the nutraceutical industry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059156PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602587PMC
September 2013

Combining integrated genomics and functional genomics to dissect the biology of a cancer-associated, aberrant transcription factor, the ASPSCR1-TFE3 fusion oncoprotein.

J Pathol 2013 Apr 5;229(5):743-754. Epub 2013 Mar 5.

Department of Pathology and Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, USA.

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1-TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1-TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1-TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1-TFE3, identified 2193 genes bound by ASPSCR1-TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1-TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1-TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1-TFE3. As validation of this approach to identify genuine ASPSCR1-TFE3 target genes, two up-regulated genes bound by ASPSCR1-TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1-TFE3. As the results indicated that ASPSCR1-TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1-TFE3 target genes contribute to the growth of ASPSCR1-TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.
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http://dx.doi.org/10.1002/path.4158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083568PMC
April 2013

An arrayed genome-scale lentiviral-enabled short hairpin RNA screen identifies lethal and rescuer gene candidates.

Assay Drug Dev Technol 2013 Apr 30;11(3):173-90. Epub 2012 Nov 30.

High-Throughput Screening Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of candidates toward a better understanding of cellular homeostasis. Using a high-stringency hit nomination method encompassing criteria of at least three active hairpins per gene and filtered for potential off-target effects (OTEs), referred to as the Bhinder-Djaballah analysis method, we identified 1,252 lethal and 6 rescuer gene candidates, knockdown of which resulted in severe cell death or enhanced growth, respectively. Cross referencing individual hairpins with the TRC1 validated clone database, 239 of the 1,252 candidates were deemed independently validated with at least three validated clones. Through our systematic OTE analysis, we have identified 31 microRNAs (miRNAs) in lethal and 2 in rescuer genes; all having a seed heptamer mimic in the corresponding shRNA hairpins and likely cause of the OTE observed in our screen, perhaps unraveling a previously unknown plausible essentiality of these miRNAs in cellular viability. Taken together, we report on a methodology for performing large-scale arrayed shRNA screens, a comprehensive analysis method to nominate high-confidence hits, and a performance assessment of the TRC1 library highlighting the intracellular inefficiencies of shRNA processing in general.
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http://dx.doi.org/10.1089/adt.2012.475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619155PMC
April 2013

A class of allosteric caspase inhibitors identified by high-throughput screening.

Mol Cell 2012 Aug 12;47(4):585-95. Epub 2012 Jul 12.

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Caspase inhibition is a promising approach for treating multiple diseases. Using a reconstituted assay and high-throughput screening, we identified a group of nonpeptide caspase inhibitors. These inhibitors share common chemical scaffolds, suggesting the same mechanism of action. They can inhibit apoptosis in various cell types induced by multiple stimuli; they can also inhibit caspase-1-mediated interleukin generation in macrophages, indicating potential anti-inflammatory application. While these compounds inhibit all the tested caspases, kinetic analysis indicates they do not compete for the catalytic sites of the enzymes. The cocrystal structure of one of these compounds with caspase-7 reveals that it binds to the dimerization interface of the caspase, another common structural element shared by all active caspases. Consistently, biochemical analysis demonstrates that the compound abates caspase-8 dimerization. Based on these kinetic, biochemical, and structural analyses, we suggest that these compounds are allosteric caspase inhibitors that function through binding to the dimerization interface of caspases.
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http://dx.doi.org/10.1016/j.molcel.2012.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428514PMC
August 2012

A high-content biosensor-based screen identifies cell-permeable activators and inhibitors of EGFR function: implications in drug discovery.

J Biomol Screen 2012 Aug 9;17(7):885-99. Epub 2012 May 9.

Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell-permeable modulators for development. For this purpose, we have used our recently optimized epidermal growth factor receptor (EGFR) biosensor assay to screen for modulators of EGFR activity. Here, we report on its validation under high-throughput screening (HTS) conditions displaying a signal-to-noise ratio of 21 and a Z' value of 0.56-attributes of a robust cell-based assay. We performed a pilot screen against a library of 6912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95%, respectively. Follow-up dose-response studies revealed that 12 of the 13 known EGFR inhibitors in the library were confirmed as hits. ZM-306416, a vascular endothelial growth factor receptor (VEGFR) antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone, and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.
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http://dx.doi.org/10.1177/1087057112446174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615554PMC
August 2012

Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.

Assay Drug Dev Technol 2012 Feb 26;10(1):24-36. Epub 2012 Jan 26.

HTS Core Facility, Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center , New York, New York 10065, USA.

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.
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http://dx.doi.org/10.1089/adt.2011.423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277729PMC
February 2012

A high throughput scintillation proximity imaging assay for protein methyltransferases.

Comb Chem High Throughput Screen 2012 Jun;15(5):359-71

Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Cener, New York, NY 10065, USA.

Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z' values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue, was found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553658PMC
http://dx.doi.org/10.2174/138620712800194468DOI Listing
June 2012

Identification of benzofuran-4,5-diones as novel and selective non-hydroxamic acid, non-peptidomimetic based inhibitors of human peptide deformylase.

Bioorg Med Chem Lett 2011 Aug 12;21(15):4528-32. Epub 2011 Jun 12.

Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their structure-activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.
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http://dx.doi.org/10.1016/j.bmcl.2011.05.129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139024PMC
August 2011

Intra-arterial and oral digoxin therapy for retinoblastoma.

Ophthalmic Genet 2011 Sep 29;32(3):147-50. Epub 2011 Mar 29.

Ophthalmic Oncology Service, Memorial Sloan-Kettering Cancer Center, New York, USA.

Purpose: Preclinical studies demonstrate that cardiac glycosides such as ouabain and digoxin have antitumor effects on retinoblastoma cells in vitro and in a xenograft murine model of retinoblastoma.

Methods: Based on these findings, we report a case of intra-arterial followed by systemic oral digoxin therapy in a patient with unilateral retinoblastoma that had failed prior intra-arterial chemotherapy.

Results: Oral administration of digoxin produced no effect, while intra-arterial digoxin therapy produced a modest but measurable response that was likely limited by the inability to achieve sustained drug concentration in the eye.

Conclusions: This case highlights both the potential promise and limitations of cardiac glycoside therapy in retinoblastoma.
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http://dx.doi.org/10.3109/13816810.2010.544530DOI Listing
September 2011

Validation of a high-content screening assay using whole-well imaging of transformed phenotypes.

Assay Drug Dev Technol 2011 Jun 23;9(3):247-61. Epub 2010 Dec 23.

Molecular Pharmacology and Chemistry Program, HTS Core Facility, Memorial Sloan-Kettering Cancer Center, New York, USA.

Automated microscopy was introduced two decades ago and has become an integral part of the discovery process as a high-content screening platform with noticeable challenges in executing cell-based assays. It would be of interest to use it to screen for reversers of a transformed cell phenotype. In this report, we present data obtained from an optimized assay that identifies compounds that reverse a transformed phenotype induced in NIH-3T3 cells by expressing a novel oncogene, KP, resulting from fusion between platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR), that was identified in human glioblastoma. Initial image acquisitions using multiple tiles per well were found to be insufficient as to accurately image and quantify the clusters; whole-well imaging, performed on the IN Cell Analyzer 2000, while still two-dimensional imaging, was found to accurately image and quantify clusters, due largely to the inherent variability of their size and well location. The resulting assay exhibited a Z' value of 0.79 and a signal-to-noise ratio of 15, and it was validated against known effectors and shown to identify only PDGFRα inhibitors, and then tested in a pilot screen against a library of 58 known inhibitors identifying mostly PDGFRα inhibitors as reversers of the KP induced transformed phenotype. In conclusion, our optimized and validated assay using whole-well imaging is robust and sensitive in identifying compounds that reverse the transformed phenotype induced by KP with a broader applicability to other cell-based assays that are challenging in HTS against chemical and RNAi libraries.
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http://dx.doi.org/10.1089/adt.2010.0342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123874PMC
June 2011

Cell viability assessment: toward content-rich platforms.

Expert Opin Drug Discov 2010 Mar 29;5(3):223-33. Epub 2010 Jan 29.

HTS Core Facility, Molecular Pharmacology & Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NY 10065, USA.

Importance Of The Field: Monitoring cell viability in vitro is critical in many areas of biomedical research, and the ultimate goal in drug discovery is the ability to predict the in vivo toxicology of drug candidates based on their toxicity profile in vitro. Over the last decade, the contribution of high-throughput screening toward this goal has been tremendous, providing the ability to screen compounds in parallel against multiple cell types. However, the toxic effects of drug candidates uncovered during clinical trials are by far the main reason for their failure. Over the same period, our understanding of programmed cell death has evolved dramatically with the identification of critical control points in the cell death pathways. As a result, cell viability should no longer be characterized solely on the basis of discrete end point measurements such as membrane permeability.

Areas Covered In This Review: This review summarizes the traditional viability assays currently commercially available, focusing on methods amenable to high density format. Assays categorized into the following classes are discussed: dye exclusion assays, DNA condensation-based assays and assays monitoring a metabolic function.

What The Reader Will Gain: We describe current approaches for assessing cell viability and, using case studies, emphasize their limitations. As an alternative, we propose the use of live, multiplexed readouts to accurately record cell death induction.

Take Home Message: Current low-content methods based on single parameter readouts are prone to error due to the heterogeneity of cell populations and the multi-faceted nature of cell death. High-content approaches based on continuous, multiplexed readouts are becoming increasingly important for monitoring multiple markers of cell death induction simultaneously on a cell by cell basis. The use of such content-rich platforms is a necessity to predict the toxicology of drug candidates accurately.
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http://dx.doi.org/10.1517/17460441003596685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3640448PMC
March 2010

Structure-activity relationships of 6-(2,6-dichlorophenyl)-8-methyl-2-(phenylamino)pyrido[2,3-d]pyrimidin-7-ones: toward selective Abl inhibitors.

Bioorg Med Chem Lett 2009 Dec 23;19(24):6872-6. Epub 2009 Oct 23.

Department of Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NY 10065, USA.

We report the design, synthesis, and structure-activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well-characterized cancer cell lines. Our study reveals that substitutions in the 3- and 4-positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell-based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.
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http://dx.doi.org/10.1016/j.bmcl.2009.10.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629380PMC
December 2009

Live-cell imaging of caspase activation for high-content screening.

J Biomol Screen 2009 Sep 2;14(8):956-69. Epub 2009 Sep 2.

High Throughput Screening Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488 fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposide, or cell death siRNA. This method was further validated against 2 well-characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to erlotinib, where the authors show a differential time-dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, the results demonstrate the feasibility of using this newly adapted and validated high-content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.
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http://dx.doi.org/10.1177/1087057109343207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613133PMC
September 2009

Development and validation of a high-density fluorescence polarization-based assay for the trypanosoma RNA triphosphatase TbCet1.

Comb Chem High Throughput Screen 2009 Mar;12(3):258-68

High Throughput Screening Core Facility, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

RNA triphosphatases are attractive and mostly unexplored therapeutic targets for the development of broad spectrum antiprotozoal, antiviral and antifungal agents. The use of malachite green as a readout for phosphatases is well characterized and widely employed. However, the reaction depends on high quantities of inorganic phosphate to be generated, which makes this assay not easily amenable to screening in 1536-well format. The overly long reading times required also prohibit its use to screen large chemical libraries. To overcome these limitations, we sought to develop a fluorescence polarization (FP) -based assay for triphosphatases, compatible with miniaturization and fast readouts. For this purpose, we took advantage of the nucleoside triphosphatase activity of this class of enzyme to successfully adapt the Transcreener ADP assay based on the detection of generated ADP by immunocompetition fluorescence polarization to the RNA triphosphatase TbCet1 in 1536-well format. We also tested the performance of this newly developed assay in a pilot screen of 3,000 compounds and we confirmed the activity of the obtained hits. We present and discuss our findings and their importance for the discovery of novel drugs by high throughput screening.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626118PMC
http://dx.doi.org/10.2174/138620709787581729DOI Listing
March 2009