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Anthony B Mak,
Allison M L Nixon,
Saranya Kittanakom,
Jocelyn M Stewart,
Ginny I Chen,
Jasna Curak,
Anne-Claude Gingras,
Ralph Mazitschek,
Benjamin G Neel,
Igor Stagljar, Jason Moffat
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ABSTRACT: The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, β-catenin, can physically associate as a ternary complex. This association stabilizes β-catenin via HDAC6 deacetylase activity, which leads to activation of β-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased β-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.
Cell reports. 10/2012;
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Johnny M Tkach,
Askar Yimit,
Anna Y Lee,
Michael Riffle,
Michael Costanzo,
Daniel Jaschob,
Jason A Hendry,
Jiongwen Ou, Jason Moffat,
Charles Boone,
Trisha N Davis,
Corey Nislow,
Grant W Brown
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ABSTRACT: Relocalization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein reorganization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by subcellular destination enables the identification of pathways that respond to replication stress. We analysed pairwise combinations of GFP fusions and gene deletion mutants to define and order two previously unknown DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways.
Nature Cell Biology 07/2012; 14(9):966-76. · 19.49 Impact Factor
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ABSTRACT: Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.
Methods 06/2012; 57(4):409-16. · 4.01 Impact Factor
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ABSTRACT: Acquisition of resistance to anchorage dependant cell death, a process termed anoikis, is a requirement for cancer cell metastasis. However, the molecular determinants of anoikis resistance and sensitivity are poorly understood. To better understand resistance to anoikis we conducted a genome wide lentiviral shRNA screen to identify genes whose knockdown render anoikis-sensitive RWPE-1 prostate cells resistant to anoikis. RWPE-1 cells were infected with a pooled lentiviral shRNA library with 54,021 shRNA targeting 11,255 genes. After infection, an anoikis-resistant cell population was selected and shRNA sequences were amplified and sequenced. Thirty-four shRNA sequences reproducibly protected RWPE-1 cells from anoikis after culture under suspension conditions including the top validated hit, α/β hydrolase domain containing 4 (ABHD4). In validation studies, ABHD4 knockdown inhibited anoikis in RWPE-1 cells as well as anoikis sensitive NP69 nasopharyngeal and OVCAR3 ovarian cancer cells, while over-expression of the gene increased sensitivity. Induction of anoikis after ABHD4 knockdown was associated with cleavage of PARP and activation of caspases-3, but was independent in changes of FLIP, FAK and Src expression. Interestingly, induction of anoikis after ABHD4 knockdown was independent of the known role of ABHD4 in the anandamide synthesis pathway and the generation of glycerophospho-N-acyl ethanolamines. Thus, ABHD4 is a novel genetic regulator of anoikis sensitivity.
Apoptosis 04/2012; 17(7):666-78. · 4.07 Impact Factor
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ABSTRACT: The AC133 epitope has been used as a marker for both normal and cancer stem cells from multiple tissue lineages. To identify transcription factors that regulate CD133 expression, we conducted parallel large-scale RNA interference screens in Caco-2 cancer cells that endogenously express CD133 and in engineered HEK293 cells that express CD133 from a heterologous promoter. The transcription factor AF4 was identified following a comparative analysis between the two screens. We then showed that AF4 is a promoter of CD133 transcription in multiple cancer cell lines. Knockdown of AF4 resulted in a dramatic reduction in CD133 transcript levels. Importantly, a subset of pediatric acute lymphoblastic leukemias (ALL) harbor a fusion oncogene results from a chromosomal translocation that juxtaposes the mixed-lineage leukemia (MLL) gene and the AF4 gene. An investigation of the functional role of CD133 in the MLL-AF4-dependent ALL cells revealed that CD133 was required for leukemia cell survival. Together, our findings show AF4-dependent regulation of CD133 expression, which is required for the growth of ALL cells. CD133 may therefore represent a therapeutic target in a subset of cancers.
Cancer Research 02/2012; 72(8):1929-34. · 7.86 Impact Factor
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Richard Marcotte,
Kevin R Brown,
Fernando Suarez,
Azin Sayad,
Konstantina Karamboulas,
Paul M Krzyzanowski,
Fabrice Sircoulomb,
Mauricio Medrano,
Yaroslav Fedyshyn,
Judice L Y Koh, [......],
Alla Buzina,
Patricia Mero,
Christine Misquitta,
Josee Normand,
Maliha Haider,
Troy Ketela,
Jeffrey L Wrana,
Robert Rottapel,
Benjamin G Neel, Jason Moffat
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ABSTRACT: Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype ("drivers"). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a "functional genomic" map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ~16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types. SIGNIFICANCE : This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. SIGNIFICANCE: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types.
Cancer discovery. 02/2012; 2(2):172-89.
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Reza Beheshti Zavareh,
Mahadeo A Sukhai,
Rose Hurren,
Marcela Gronda,
Xiaoming Wang,
Craig D Simpson,
Neil Maclean,
Francis Zih,
Troy Ketela,
Carol J Swallow, Jason Moffat,
David R Rose,
Harry Schachter,
Aaron D Schimmer,
James W Dennis
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ABSTRACT: Oncogenic signaling promotes tumor invasion and metastasis, in part, by increasing the expression of tri- and tetra- branched N-glycans. The branched N-glycans bind to galectins forming a multivalent lattice that enhances cell surface residency of growth factor receptors, and focal adhesion turnover. N-acetylglucosaminyltransferase I (MGAT1), the first branching enzyme in the pathway, is required for the addition of all subsequent branches. Here we have introduced MGAT1 shRNA into human HeLa cervical and PC-3-Yellow prostate tumor cells lines, generating cell lines with reduced transcript, enzyme activity and branched N-glycans at the cell surface. MGAT1 knockdown inhibited HeLa cell migration and invasion, but did not alter cell proliferation rates. Swainsonine, an inhibitor of α-mannosidase II immediately downstream of MGAT1, also inhibited cell invasion and was not additive with MGAT1 shRNA, consistent with a common mechanism of action. Focal adhesion and microfilament organization in MGAT1 knockdown cells also indicate a less motile phenotype. In vivo, MGAT1 knockdown in the PC-3-Yellow orthotopic prostate cancer xenograft model significantly decreased primary tumor growth and the incidence of lung metastases. Our results demonstrate that blocking MGAT1 is a potential target for anti-cancer therapy.
PLoS ONE 01/2012; 7(9):e43721. · 4.09 Impact Factor
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ABSTRACT: Genome-wide pooled shRNA screens enable global identification of the genes essential for cancer cell survival and proliferation and provide a 'functional genetic' map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting approximately 16,000 human genes and a newly developed scoring approach, we identified essential gene profiles in more than 70 breast, pancreatic and ovarian cancer cell lines. We developed a web-accessible database system for capturing information from each step in our standardized screening pipeline and a gene-centric search tool for exploring shRNA activities within a given cell line or across multiple cell lines. The database consists of a laboratory information and management system for tracking each step of a pooled shRNA screen as well as a web interface for querying and visualization of shRNA and gene-level performance across multiple cancer cell lines. COLT-Cancer Version 1.0 is currently accessible at http://colt.ccbr.utoronto.ca/cancer.
Nucleic Acids Research 11/2011; 40(Database issue):D957-63. · 8.03 Impact Factor
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Marko Skrtić,
Shrivani Sriskanthadevan,
Bozhena Jhas,
Marinella Gebbia,
Xiaoming Wang,
Zezhou Wang,
Rose Hurren,
Yulia Jitkova,
Marcela Gronda,
Neil Maclean, [......],
Jessie H Cameron,
Jeffery Wrana,
Connie J Eaves,
Mark D Minden,
Jean C Y Wang,
John E Dick,
Keith Humphries,
Corey Nislow,
Guri Giaever,
Aaron D Schimmer
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ABSTRACT: To identify FDA-approved agents targeting leukemic cells, we performed a chemical screen on two human leukemic cell lines and identified the antimicrobial tigecycline. A genome-wide screen in yeast identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated lethality. Tigecycline selectively killed leukemia stem and progenitor cells compared to their normal counterparts and also showed antileukemic activity in mouse models of human leukemia. ShRNA-mediated knockdown of EF-Tu mitochondrial translation factor in leukemic cells reproduced the antileukemia activity of tigecycline. These effects were derivative of mitochondrial biogenesis that, together with an increased basal oxygen consumption, proved to be enhanced in AML versus normal hematopoietic cells and were also important for their difference in tigecycline sensitivity.
Cancer cell 11/2011; 20(5):674-88. · 25.29 Impact Factor
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ABSTRACT: The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.
Journal of Biological Chemistry 09/2011; 286(47):41046-56. · 4.77 Impact Factor
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Cell 09/2011; 146(6):1044, 1044.e1-2. · 32.40 Impact Factor
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ABSTRACT: Evasion of death receptor ligand-induced apoptosis contributs to cancer development and progression. To better understand mechanisms conferring resistance to death ligands, we screened an siRNA library to identify sequences that sensitize resistant cells to fas activating antibody (CH-11). From this screen, we identified the Sterol-Regulatory Element-Binding Protein 1 (SREBP1), a transcription factor, which regulates genes involved in cholesterol and fatty acid synthesis including fatty acid synthase. Inhibition of SREBP1 sensitized PPC-1 and HeLa to the death receptor ligands CH-11 and TRAIL. In contrast, DU145 prostate cancer cells that are resistant to death ligands despite expressing the receptors on their cell surface remained resistant to CH-11 and TRAIL after knockdown of SREBP1. Consistent with the effects on cell viability, the addition of CH-11 activated caspases 3 and 8 in HeLa but not DU145 cells with silenced SREBP1. We demonstrated that knockdown of SREBP1 produced a marked decrease in fatty acid synthase expression. Furthermore, genetic or chemical inhibition of fatty acid synthase with shRNA or orlistat, respectively, recapitulated the effects of SREBP1 inhibition and sensitized HeLa but not DU145 cells to CH-11 and TRAIL. Sensitization to death receptor ligands by inhibition of fatty acid synthase was associated with activation of caspase 8 prior to caspase 9. Neither silencing of SREBP1 or fatty acid synthase changed basal expression of the core death receptor components Fas, caspase 8, FADD, caspase 3 or FLIP. Thus, inhibition of SREBP1 or its downstream target fatty acid synthase sensitizes resistant cells to death ligands.
Oncotarget 03/2011; 2(3):186-96. · 4.78 Impact Factor
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ABSTRACT: Genome sequencing efforts have reformed the nature of biological inquiry, prompting the development of technologies for the functional annotation of mammalian genes. Based on methodologies originally discovered in plants and Caenorhabditis elegans, RNA interference has offered cell biologists an effective and reproducible approach to perturb gene function in mammalian cells and whole organisms. Initial application of RNA interference libraries targeting the human and mouse genomes relied on arrayed screening approaches, whereby each unique RNA interference reagent is arrayed into individual wells of a microtiter plate. These screens are not trivial to perform, requiring a substantial investment in infrastructure. In the past decade, many technological advances have been made that make genome-wide RNA interference screening more accessible to researchers and more feasible to perform in nonspecialized laboratories. Here, we describe a comprehensive protocol for pooled short-hairpin RNA screening, including methodologies for pooled lentivirus production, cell infection, genome-wide negative selection screening and resources for pooled screen deconvolution, and data analysis. As a technique, pooled shRNA screening is still in its infancy, but the methodology has already been successfully applied to probe diverse signaling pathways, as a means of drug target identification, and to identify essential genes in normal and cancer cell lines.
Methods in molecular biology (Clifton, N.J.) 01/2011; 781:161-82.
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Troy Ketela,
Lawrence E Heisler,
Kevin R Brown,
Ron Ammar,
Dahlia Kasimer,
Anuradha Surendra,
Elke Ericson,
Kim Blakely,
Dina Karamboulas,
Andrew M Smith, [......],
Kahlin Cheung-Ong,
Judice L Y Koh,
Shuba Gopal,
Glenn S Cowley,
Xiaoping Yang,
Jennifer K Grenier,
Guri Giaever,
David E Root, Jason Moffat,
Corey Nislow
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ABSTRACT: Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens.
Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens.
Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.
BMC Genomics 01/2011; 12:213. · 4.07 Impact Factor
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Jacky Chung,
Joanne Lau,
Lynn S Cheng,
R Ian Grant,
Fiona Robinson,
Troy Ketela,
Patricia P Reis,
Olga Roche,
Suzanne Kamel-Reid, Jason Moffat,
Michael Ohh,
Bayardo Perez-Ordonez,
David R Kaplan,
Meredith S Irwin
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ABSTRACT: ΔNp63α is a critical pro-survival protein overexpressed in 80% of head and neck squamous cell carcinomas (HNSCCs) where it inhibits TAp73β transcription of p53-family target genes, which is thought to increase HNSCC resistance to chemotherapy-induced cell death. However, the mechanisms governing ΔNp63α function are largely unknown. In this study, we identify special AT-rich-binding protein 2 (SATB2) as a new ΔNp63α-binding protein that is preferentially expressed in advanced-stage primary HNSCC and show that SATB2 promotes chemoresistance by enhancing ΔNp63α-mediated transrepression by augmenting ΔNp63α engagement to p53-family responsive elements. Furthermore, SATB2 expression positively correlates with HNSCC chemoresistance, and RNA interference-mediated knockdown of endogenous SATB2 re-sensitizes HNSCC cells to chemotherapy- and γ-irradiation-induced apoptosis, irrespective of p53 status. These findings unveil SATB2 as a pivotal modulator of ΔNp63α that governs HNSCC cell survival.
EMBO Reports 10/2010; 11(10):777-83. · 7.36 Impact Factor
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Olena Morozova,
Milijana Vojvodic,
Natalie Grinshtein,
Loen M Hansford,
Kim M Blakely,
Alexandra Maslova,
Martin Hirst,
Timothee Cezard,
Ryan D Morin,
Richard Moore, [......],
Paul Taylor,
Nina Thiessen,
Richard Varhol,
Yongjun Zhao,
Steven Jones, Jason Moffat,
Thomas Kislinger,
Michael F Moran,
David R Kaplan,
Marco A Marra
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ABSTRACT: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences.
We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result.
Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells.
This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets.
Clinical Cancer Research 09/2010; 16(18):4572-82. · 7.74 Impact Factor
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Kristen M Smith,
Alessandro Datti,
Mayumi Fujitani,
Natalie Grinshtein,
Libo Zhang,
Olena Morozova,
Kim M Blakely,
Susan A Rotenberg,
Loen M Hansford,
Freda D Miller,
Herman Yeger,
Meredith S Irwin, Jason Moffat,
Marco A Marra,
Sylvain Baruchel,
Jeffrey L Wrana,
David R Kaplan
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ABSTRACT: Neuroblastoma (NB) is the most deadly extra-cranial solid tumour in children necessitating an urgent need for effective and less toxic treatments. One reason for the lack of efficacious treatments may be the inability of existing drugs to target the tumour-initiating or cancer stem cell population responsible for sustaining tumour growth, metastases and relapse. Here, we describe a strategy to identify compounds that selectively target patient-derived cancer stem cell-like tumour-initiating cells (TICs) while sparing normal paediatric stem cells (skin-derived precursors, SKPs) and characterize two therapeutic candidates. DECA-14 and rapamycin were identified as NB TIC-selective agents. Both compounds induced TIC death at nanomolar concentrations in vitro, significantly reduced NB xenograft tumour weight in vivo, and dramatically decreased self-renewal or tumour-initiation capacity in treated tumours. These results demonstrate that differential drug sensitivities between TICs and normal paediatric stem cells can be exploited to identify novel, patient-specific and potentially less toxic therapies.
EMBO Molecular Medicine 09/2010; 2(9):371-84. · 10.33 Impact Factor
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G Wei Xu,
Mohsin Ali,
Tabitha E Wood,
Derek Wong,
Neil Maclean,
Xiaoming Wang,
Marcela Gronda,
Marko Skrtic,
Xiaoming Li,
Rose Hurren,
Xinliang Mao,
Meenakshi Venkatesan,
Reza Beheshti Zavareh,
Troy Ketela,
John C Reed,
David Rose, Jason Moffat,
Robert A Batey,
Sirano Dhe-Paganon,
Aaron D Schimmer
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ABSTRACT: The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.
Blood 03/2010; 115(11):2251-9. · 9.90 Impact Factor
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Anthony B Mak,
Zuyao Ni,
Johannes A Hewel,
Ginny I Chen,
Guoqing Zhong,
Konstantina Karamboulas,
Kim Blakely,
Sandra Smiley,
Edyta Marcon,
Denitza Roudeva,
Joyce Li,
Jonathan B Olsen,
Cuihong Wan,
Thanuja Punna,
Ruth Isserlin,
Sergei Chetyrkin,
Anne-Claude Gingras,
Andrew Emili,
Jack Greenblatt, Jason Moffat
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ABSTRACT: Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.
Molecular & Cellular Proteomics 03/2010; 9(5):811-23. · 7.40 Impact Factor
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Tabitha E Wood,
Shadi Dalili,
Craig D Simpson,
Mahadeo A Sukhai,
Rose Hurren,
Kika Anyiwe,
Xinliang Mao,
Fernando Suarez Saiz,
Marcela Gronda,
Yanina Eberhard,
Neil MacLean,
Troy Ketela,
John C Reed, Jason Moffat,
Mark D Minden,
Robert A Batey,
Aaron D Schimmer
[show abstract]
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ABSTRACT: Evasion of death receptor ligand-induced apoptosis represents an important contributor to cancer development and progression. Therefore, molecules that restore sensitivity to death receptor stimuli would be important tools to better understand this biological pathway and potential leads for therapeutic adjuncts. Previously, the small-molecule 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (that we propose be named droxinostat) was identified as a chemical sensitizer to death receptor stimuli, decreasing the expression of the caspase-8 inhibitor FLIP. However, the direct targets of droxinostat were unknown. To better understand the mechanism of action of droxinostat and highlight new strategies to restore sensitivity to death receptor ligands, we analyzed changes in gene expression using the Connectivity Map after treating cells with droxinostat. Changes in gene expression after droxinostat treatment resembled changes observed after treatment with histone deacetylase (HDAC) inhibitors. Therefore, we examined the effects of droxinostat on HDAC activity and showed that it selectively inhibited HDAC3, HDAC6, and HDAC8 and that inhibition of these HDACs was functionally important for its ability to sensitize cells to death ligands. Thus, we have identified a selective HDAC inhibitor and showed that selective HDAC inhibition sensitizes cells to death ligands, thereby highlighting a new mechanism to overcome resistance to death receptor ligands.
Molecular Cancer Therapeutics 01/2010; 9(1):246-56. · 5.23 Impact Factor
