Publications by authors named "Frederic Sigoillot"

28 Publications

  • Page 1 of 1

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Cell Chem Biol 2021 Mar 30. Epub 2021 Mar 30.

Novartis Institutes for BioMedical Research, 181 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.
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http://dx.doi.org/10.1016/j.chembiol.2021.03.004DOI Listing
March 2021

Benchmarking network algorithms for contextualizing genes of interest.

PLoS Comput Biol 2019 12 20;15(12):e1007403. Epub 2019 Dec 20.

Respiratory Disease Area Department, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.

Computational approaches have shown promise in contextualizing genes of interest with known molecular interactions. In this work, we evaluate seventeen previously published algorithms based on characteristics of their output and their performance in three tasks: cross validation, prediction of drug targets, and behavior with random input. Our work highlights strengths and weaknesses of each algorithm and results in a recommendation of algorithms best suited for performing different tasks.
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http://dx.doi.org/10.1371/journal.pcbi.1007403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944391PMC
December 2019

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

Nat Chem Biol 2020 01 9;16(1):50-59. Epub 2019 Dec 9.

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

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
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http://dx.doi.org/10.1038/s41589-019-0424-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116157PMC
January 2020

A Genome-wide CRISPR Screen Identifies ZCCHC14 as a Host Factor Required for Hepatitis B Surface Antigen Production.

Cell Rep 2019 Dec;29(10):2970-2978.e6

Novartis Institutes for BioMedical Research, Emeryville, CA 94608, USA.

A hallmark of chronic hepatitis B (CHB) virus infection is the presence of high circulating levels of non-infectious small lipid HBV surface antigen (HBsAg) vesicles. Although rare, sustained HBsAg loss is the idealized endpoint of any CHB therapy. A small molecule, RG7834, has been previously reported to inhibit HBsAg expression by targeting terminal nucleotidyltransferase proteins 4A and 4B (TENT4A and TENT4B). In this study, we describe a genome-wide CRISPR screen to identify other potential host factors required for HBsAg expression and to gain further insights into the mechanism of RG7834. We report more than 60 genes involved in regulating HBsAg and identify additional factors involved in RG7834 activity, including a zinc finger CCHC-type containing 14 (ZCCHC14) protein. We show that ZCCHC14, together with TENT4A/B, stabilizes HBsAg expression through HBV RNA tailing, providing a potential new therapeutic target to achieve functional cure in CHB patients.
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http://dx.doi.org/10.1016/j.celrep.2019.10.113DOI Listing
December 2019

Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.

Elife 2019 11 19;8. Epub 2019 Nov 19.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, United States.

EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of , essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from knockout. We also show that knockout of , or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.
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http://dx.doi.org/10.7554/eLife.50223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927754PMC
November 2019

USP7 inhibits Wnt/β-catenin signaling through promoting stabilization of Axin.

Nat Commun 2019 09 13;10(1):4184. Epub 2019 Sep 13.

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

Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.
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http://dx.doi.org/10.1038/s41467-019-12143-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744515PMC
September 2019

YAP, but Not RSPO-LGR4/5, Signaling in Biliary Epithelial Cells Promotes a Ductular Reaction in Response to Liver Injury.

Cell Stem Cell 2019 07 9;25(1):39-53.e10. Epub 2019 May 9.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland. Electronic address:

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/β-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.
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http://dx.doi.org/10.1016/j.stem.2019.04.005DOI Listing
July 2019

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

Hyperactivation of MAPK Signaling Is Deleterious to RAS/RAF-mutant Melanoma.

Mol Cancer Res 2019 01 10;17(1):199-211. Epub 2018 Sep 10.

Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF-MEK-ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAF-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAF melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation-induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against and rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. IMPLICATIONS: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0327DOI Listing
January 2019

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C.

J Clin Invest 2017 12 13;127(12):4554-4568. Epub 2017 Nov 13.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Transcriptional repression of ubiquitin B (UBB) is a cancer-subtype-specific alteration that occurs in a substantial population of patients with cancers of the female reproductive tract. UBB is 1 of 2 genes encoding for ubiquitin as a polyprotein consisting of multiple copies of ubiquitin monomers. Silencing of UBB reduces cellular UBB levels and results in an exquisite dependence on ubiquitin C (UBC), the second polyubiquitin gene. UBB is repressed in approximately 30% of high-grade serous ovarian cancer (HGSOC) patients and is a recurrent lesion in uterine carcinosarcoma and endometrial carcinoma. We identified ovarian tumor cell lines that retain UBB in a repressed state, used these cell lines to establish orthotopic ovarian tumors, and found that inducible expression of a UBC-targeting shRNA led to tumor regression, and substantial long-term survival benefit. Thus, we describe a recurrent cancer-specific lesion at the level of ubiquitin production. Moreover, these observations reveal the prognostic value of UBB repression and establish UBC as a promising therapeutic target for ovarian cancer patients with recurrent UBB silencing.
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http://dx.doi.org/10.1172/JCI92914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707153PMC
December 2017

Explicit Modeling of siRNA-Dependent On- and Off-Target Repression Improves the Interpretation of Screening Results.

Cell Syst 2017 02 15;4(2):182-193.e4. Epub 2017 Feb 15.

Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. Electronic address:

RNAi is broadly used to map gene regulatory networks, but the identification of genes that are responsible for the observed phenotypes is challenging, as small interfering RNAs (siRNAs) simultaneously downregulate the intended on targets and many partially complementary off targets. Additionally, the scarcity of publicly available control datasets hinders the development and comparative evaluation of computational methods for analyzing the data. Here, we introduce PheLiM (https://github.com/andreariba/PheLiM), a method that uses predictions of siRNA on- and off-target downregulation to infer gene-specific contributions to phenotypes. To assess the performance of PheLiM, we carried out siRNA- and CRISPR/Cas9-based genome-wide screening of two well-characterized pathways, bone morphogenetic protein (BMP) and nuclear factor κB (NF-κB), and we reanalyzed publicly available siRNA screens. We demonstrate that PheLiM has the overall highest accuracy and most reproducible results compared to other available methods. PheLiM can accommodate various methods for predicting siRNA off targets and is broadly applicable to the identification of genes underlying complex phenotypes.
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http://dx.doi.org/10.1016/j.cels.2017.01.011DOI Listing
February 2017

MiR-210 promotes sensory hair cell formation in the organ of corti.

BMC Genomics 2016 Apr 27;17:309. Epub 2016 Apr 27.

Developmental and Molecular Pathways, Novartis Institute for Biomedical Research, Basel, Switzerland.

Background: Hearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss.

Results: In order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach.

Conclusion: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.
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http://dx.doi.org/10.1186/s12864-016-2620-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848794PMC
April 2016

Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C.

Nature 2014 Oct 24;514(7524):646-9. Epub 2014 Aug 24.

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the anaphase-promoting complex/cyclosome (APC/C), a 13-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome. Because blocking mitotic exit is an effective approach for inducing tumour cell death, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc20 (ref. 5), which forms a co-receptor with the APC/C to recognize substrates containing a destruction box (D-box). Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identify a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-l-arginine methyl ester, a small molecule that blocks the APC/C-Cdc20 interaction. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine.
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http://dx.doi.org/10.1038/nature13660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4214887PMC
October 2014

Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis.

BMC Bioinformatics 2014 Jun 17;15:192. Epub 2014 Jun 17.

Drosophila RNAi Screening Center, Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

Background: RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial.

Results: Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence matches (GESS) was developed to identify potential off-targeted transcripts in large-scale screen data by seed-region analysis. Here, we introduce a user-friendly web application that provides researchers a relatively quick and easy way to perform GESS analysis on data from human or mouse cell-based screens using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), as well as for Drosophila screens using shRNAs. Online GESS relies on up-to-date transcript sequence annotations for human and mouse genes extracted from NCBI Reference Sequence (RefSeq) and Drosophila genes from FlyBase. The tool also accommodates analysis with user-provided reference sequence files.

Conclusion: Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences.
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http://dx.doi.org/10.1186/1471-2105-15-192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073188PMC
June 2014

The G2/M regulator histone demethylase PHF8 is targeted for degradation by the anaphase-promoting complex containing CDC20.

Mol Cell Biol 2013 Nov 26;33(21):4166-80. Epub 2013 Aug 26.

Division of Newborn Medicine and Program in Epigenetics, Department of Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.

Monomethylated histone H4 lysine 20 (H4K20me1) is tightly regulated during the cell cycle. The H4K20me1 demethylase PHF8 transcriptionally regulates many cell cycle genes and is therefore predicted to play key roles in the cell cycle. Here, we show that PHF8 protein levels are the highest during G2 phase and mitosis, and we found PHF8 protein stability to be regulated by the ubiquitin-proteasome system. Purification of the PHF8 complex led to the identification of many subunits of the anaphase-promoting complex (APC) associated with PHF8. We showed that PHF8 interacts with the CDC20-containing APC (APC(cdc20)) primarily during mitosis. In addition, we defined a novel, KEN- and D-box-independent, LXPKXLF motif on PHF8 that is required for binding to CDC20. Through various in vivo and in vitro assays, we demonstrate that mutations of the LXPKXLF motif abrogate polyubiquitylation of PHF8 by the APC. APC substrates are typically cell cycle regulators, and consistent with this, the loss of PHF8 leads to prolonged G2 phase and defective mitosis. Furthermore, we provide evidence that PHF8 plays an important role in transcriptional activation of key G2/M genes during G2 phase. Taken together, these findings suggest that PHF8 is regulated by APC(cdc20) and plays an important role in the G2/M transition.
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http://dx.doi.org/10.1128/MCB.00689-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811896PMC
November 2013

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response.

Nat Cell Biol 2012 Feb 19;14(3):318-28. Epub 2012 Feb 19.

Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.
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http://dx.doi.org/10.1038/ncb2426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290715PMC
February 2012

A bioinformatics method identifies prominent off-targeted transcripts in RNAi screens.

Nat Methods 2012 Feb 19;9(4):363-6. Epub 2012 Feb 19.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

Because off-target effects hamper interpretation and validation of RNAi screen data, we developed a bioinformatics method, genome-wide enrichment of seed sequence matches (GESS), to identify candidate off-targeted transcripts in primary screening data. GESS analysis revealed a prominent off-targeted transcript in several screens, including MAD2 (MAD2L1) in a screen for genes required for the spindle assembly checkpoint. GESS analysis results can enhance the validation rate in RNAi screens.
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http://dx.doi.org/10.1038/nmeth.1898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3482495PMC
February 2012

A time-series method for automated measurement of changes in mitotic and interphase duration from time-lapse movies.

PLoS One 2011 26;6(9):e25511. Epub 2011 Sep 26.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, United States of America.

Background: Automated time-lapse microscopy can visualize proliferation of large numbers of individual cells, enabling accurate measurement of the frequency of cell division and the duration of interphase and mitosis. However, extraction of quantitative information by manual inspection of time-lapse movies is too time-consuming to be useful for analysis of large experiments.

Methodology/principal Findings: Here we present an automated time-series approach that can measure changes in the duration of mitosis and interphase in individual cells expressing fluorescent histone 2B. The approach requires analysis of only 2 features, nuclear area and average intensity. Compared to supervised learning approaches, this method reduces processing time and does not require generation of training data sets. We demonstrate that this method is as sensitive as manual analysis in identifying small changes in interphase or mitotic duration induced by drug or siRNA treatment.

Conclusions/significance: This approach should facilitate automated analysis of high-throughput time-lapse data sets to identify small molecules or gene products that influence timing of cell division.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025511PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180452PMC
February 2012

Vigilance and validation: Keys to success in RNAi screening.

ACS Chem Biol 2011 Jan 28;6(1):47-60. Epub 2010 Dec 28.

Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, United States.

In the 12 years since the process of RNA interference (RNAi) was first discovered, great progress has been made in understanding its mechanism and exploiting its ability to silence gene expression to study gene function at a genome-wide level. Its extensive use as a screening method has yielded many published lists of genes that play novel roles in higher eukaryotes. However, the usefulness of this information is potentially limited by the occurrence of unintended off-target effects. Here we review the potential causes of off-target effects and the impact of this phenomenon in interpreting the results of high-throughput RNAi screens. In addition to targeting the intended gene product, artificial short interfering RNAs (siRNAs) can produce off-target effects by down-regulating the expression of multiple mRNAs through microRNA-like targeting of the 3' untranslated region. We examine why this phenomenon can produce high hit rates in siRNA screens and why independent validation of screening results is critical for the approach to yield new biological insights.
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http://dx.doi.org/10.1021/cb100358fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306249PMC
January 2011

Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage.

Cancer Cell 2010 Oct;18(4):382-95

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Microtubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest.
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http://dx.doi.org/10.1016/j.ccr.2010.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957475PMC
October 2010

Protein kinase C modulates the up-regulation of the pyrimidine biosynthetic complex, CAD, by MAP kinase.

Front Biosci 2007 May 1;12:3892-8. Epub 2007 May 1.

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

The multifunctional protein CAD initiates de novo pyrimidine biosynthesis in mammalian cells. CAD is activated by MAP kinase (Erk1/2) just prior to the S phase of the cell cycle, when the demand for pyrimidine nucleotides is greatest, and down-regulated as the cells emerge from S phase by protein kinase A (PKA) phosphorylation. MAP kinase phosphorylates Thr456, while PKA phosphorylates Ser1406 and Ser1859, although only Ser1406 is involved in regulation. LC/mass spectrometry showed that Ser1873, a residue that lies within a putative protein kinase C (PKC) consensus sequence is also phosphorylated. Purified CAD was reacted with ATP and a panel of eight PKC isozymes. Most isozymes resulted in limited CAD phosphorylation, but the delta and epsilon isozymes were most effective. While the level of Thr456 phosphorylation is very low in confluent cells, exposure of stationary BHK 165-23 cells to the PKC activator, phorbol 12-myristate-13-acetate (PMA) resulted in a 3-fold increase in the modification of this residue. The stimulation of Thr456 phosphorylation was blocked by PKC inhibitors. The PKA inhibitor, H-89, also stimulated PMA-induced Thr456 modification probably because PKA mediated phosphorylation of CAD Ser1406 antagonizes the MAP kinase phosphorylation. Thus, the extent of Thr456 phosphorylation and the activation of pyrimidine biosynthesis depend on the synergistic and antagonistic interactions of three signaling pathways, MAP kinase, PKC and PKA. Deletions mutants lacking the putative PKC site, Ser1873 do not exhibit PMA induced Thr456 phosphorylation. We conclude that the activating MAP kinase phosphorylation of CAD proceeds through a PKC dependent pathway.
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http://dx.doi.org/10.2741/2358DOI Listing
May 2007

Protein kinase A phosphorylation of the multifunctional protein CAD antagonizes activation by the MAP kinase cascade.

Mol Cell Biochem 2007 Jul 6;301(1-2):69-81. Epub 2007 Jan 6.

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

The flux through the de novo pyrimidine biosynthetic pathway is controlled by the multifunctional protein CAD, which catalyzes the first three steps. The cell cycle dependent regulation of pyrimidine biosynthesis is a consequence of sequential phosphorylation of CAD Thr456 and Ser1406 by the MAP kinase and PKA cascades, respectively. Coordinated regulation of the pathway requires precise timing of the two phosphorylation events. These studies show that phosphorylation of purified CAD by PKA antagonizes MAP kinase phosphorylation, and vice versa. Similar results were observed in vivo. Forskolin activation of PKA in BHK-21 cells resulted in a 8.5 fold increase in Ser1406 phosphorylation and severely curtailed the MAP kinase mediated phosphorylation of CAD Thr456. Moreover, the relative activity of MAP kinase and PKA was found to determine the extent of Thr456 phosphorylation. Transfectants expressing elevated levels of MAP kinase resulted in a 11-fold increase in Thr456 phosphorylation, whereas transfectants that overexpress PKA reduced Thr456 phosphorylation 5-fold. While phosphorylation of one site by one kinase may induce conformational changes that interfere with phosphorylation by the other, the observation that both MAP kinase and PKA form stable complexes with CAD suggest that the mutual antagonism is the result of steric interference by the bound kinases. The reciprocal antagonism of CAD phosphorylation by MAP kinase and PKA provides an elegant mechanism to coordinate the cell cycle-dependent regulation of pyrimidine biosynthesis ensuring that signals for up- and down-regulation of the pathway do not conflict.
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http://dx.doi.org/10.1007/s11010-006-9398-xDOI Listing
July 2007

Nuclear localization and mitogen-activated protein kinase phosphorylation of the multifunctional protein CAD.

J Biol Chem 2005 Jul 12;280(27):25611-20. Epub 2005 May 12.

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

CAD is a multifunctional protein that initiates and regulates mammalian de novo pyrimidine biosynthesis. The activation of the pathway required for cell proliferation is a consequence of the phosphorylation of CAD Thr-456 by mitogen-activated protein (MAP) kinase. Although most of the CAD in the cell was cytosolic, cell fractionation and fluorescence microscopy showed that Thr(P)-456 CAD was primarily localized within the nucleus in association with insoluble nuclear substructures, including the nuclear matrix. CAD in resting cells was cytosolic and unphosphorylated. Upon epidermal growth factor stimulation, CAD moved to the nucleus, and Thr-456 was found to be phosphorylated. Mutation of the CAD Thr-456 and inhibitor studies showed that nuclear import is not mediated by MAP kinase phosphorylation. Two fluorescent CAD constructs, NLS-CAD and NES-CAD, were prepared that incorporated strong nuclear import and export signals, respectively. NLS-CAD was exclusively nuclear and extensively phosphorylated. In contrast, NES-CAD was confined to the cytoplasm, and Thr-456 remained unphosphorylated. Although alternative explanations can be envisioned, it is likely that phosphorylation occurs within the nucleus where much of the activated MAP kinase is localized. Trapping CAD in the nucleus had a minimal effect on pyrimidine metabolism. In contrast, when CAD was excluded from the nucleus, the rate of pyrimidine biosynthesis, the nucleotide pools, and the growth rate were reduced by 21, 36, and 60%, respectively. Thus, the nuclear import of CAD appears to promote optimal cell growth. UMP synthase, the bifunctional protein that catalyzes the last two steps in the pathway, was also found in both the cytoplasm and nucleus.
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http://dx.doi.org/10.1074/jbc.M504581200DOI Listing
July 2005

Breakdown of the regulatory control of pyrimidine biosynthesis in human breast cancer cells.

Int J Cancer 2004 Apr;109(4):491-8

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

The activity of the de novo pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while pyrimidine biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of pyrimidine biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P approximately Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P approximately Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P approximately Thr456, increased PKA phosphorylation and decreased pyrimidine biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P approximately Thr456 and the pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated pyrimidine biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations.
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http://dx.doi.org/10.1002/ijc.11717DOI Listing
April 2004

Cell cycle-dependent regulation of pyrimidine biosynthesis.

J Biol Chem 2003 Jan 15;278(5):3403-9. Epub 2002 Nov 15.

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201,USA.

De novo pyrimidine biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be up-regulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian pyrimidine biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G(1), the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G(1). Once activated, pyrimidine biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of pyrimidine biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.
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http://dx.doi.org/10.1074/jbc.M211078200DOI Listing
January 2003

Autophosphorylation of the mammalian multifunctional protein that initiates de novo pyrimidine biosynthesis.

J Biol Chem 2002 Jul 1;277(27):24809-17. Epub 2002 May 1.

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.
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http://dx.doi.org/10.1074/jbc.M203512200DOI Listing
July 2002

Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades.

J Biol Chem 2002 May 28;277(18):15745-51. Epub 2002 Feb 28.

Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.
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http://dx.doi.org/10.1074/jbc.M201112200DOI Listing
May 2002