- Cancer Discov. 2012 Feb;2(2):172-89. Epub 2011 Dec 29.
Essential gene profiles in breast, pancreas and ovarian cancer cells
Marcotte R, Brown KR, Suarez F, Sayad A, Karamboulas K, Krzyzanowski PM, Sircoulomb F, Medrano M, Fedyshyn Y, Koh JL, van Dyk D, Fedyshyn B, Luhova M, Brito GC, Vizeacoumar FJ, Vizeacoumar FS, Datti A, Kasimer D, Buzina A, Mero P, Misquitta C, Normand J, Haider M, Ketela T, Wrana JL, Rottapel R, Neel BG, Moffat J.
Donnelly Centre and Banting & Best Department of Medical Research, Departments of Molecular Genetics and Medical Biophysics, University of Toronto; Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network; Samuel Lunenfeld Research Institute; Division of Rheumatology, Department of Medicine, St. Michael's Hospital, Toronto, Canada; Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.
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 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. - Cell. 2011 Dec 9;147(6):1340-54.
Structural basis and sequence rules for substrate recognition by tankyrase explain the basis for cherubism disease.
Guettler S, Larose J, Petsalaki E, Gish G, Scotter A, Pawson T, Rottapel R, Sicheri F.
Centre for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8, Canada.
The poly(ADP-ribose)polymerases Tankyrase 1/2 (TNKS/TNKS2) catalyze the covalent linkage of ADP-ribose polymer chains onto target proteins, regulating their ubiquitylation, stability, and function. Dysregulation of substrate recognition by Tankyrases underlies the human disease cherubism. Tankyrases recruit specific motifs (often called RxxPDG "hexapeptides") in their substrates via an N-terminal region of ankyrin repeats. These ankyrin repeats form five domains termed ankyrin repeat clusters (ARCs), each predicted to bind substrate. Here we report crystal structures of a representative ARC of TNKS2 bound to targeting peptides from six substrates. Using a solution-based peptide library screen, we derive a rule-based consensus for Tankyrase substrates common to four functionally conserved ARCs. This 8-residue consensus allows us to rationalize all known Tankyrase substrates and explains the basis for cherubism-causing mutations in the Tankyrase substrate 3BP2. Structural and sequence information allows us to also predict and validate other Tankyrase targets, including Disc1, Striatin, Fat4, RAD54, BCR, and MERIT40. - Blood. 2012 Feb 2;119(5):1200-7. Epub 2011 Dec 9.
A small molecule screening strategy with validation on human leukemia stem cells uncovers the therapeutic efficacy of kinetin riboside.
McDermott SP, Eppert K, Notta F, Isaac M, Datti A, Al-Awar R, Wrana J, Minden MD, Dick JE.
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada;
Gene regulatory networks that govern hematopoietic stem cells (HSC) and leukemia-initiating cells (L-IC) are deeply entangled. Thus, the discovery of compounds that target L-IC while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPC). Here, we present a multi-step in vitro and in vivo approach to identify compounds that can target L-IC in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetic riboside (KR), on AML L-IC and HSPC. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC-enriched CD34+CD38- AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-IC in two of four primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti-L-IC compounds for human leukemias. - Cancer Cell. 2011 Nov 15;20(5):674-88.
Inhibition of mitochondrial translation as a therapeutic strategy for human acute myeloid leukemia.
Skrtic M, Sriskanthadevan S, Jhas B, Gebbia M, Wang X, Wang Z, Hurren R, Jitkova Y, Gronda M, Maclean N, Lai CK, Eberhard Y, Bartoszko J, Spagnuolo P, Rutledge AC, Datti A, Ketela T, Moffat J, Robinson BH, Cameron JH, Wrana J, Eaves CJ, Minden MD, Wang JC, Dick JE, Humphries K, Nislow C, Giaever G, Schimmer AD.
Campbell Family Cancer Research Institute, Princess Margaret Hospital, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada.
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. - Nucleic Acids Res. 2012 Jan;40(Database issue):D687-94. Epub 2011 Oct 18.
PhenoM: a database of morphological phenotypes caused by mutation of essential genes in Saccharomyces cerevisiae.
Jin K, Li J, Vizeacoumar FS, Li Z, Min R, Zamparo L, Vizeacoumar FJ, Datti A, Andrews B, Boone C, Zhang Z.
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON, M5S 3E1, Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, ON, M5G 1L6, Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, M5G 1X5, Canada, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122, Perugia, Italy and Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.
About one-fifth of the genes in the budding yeast are essential for haploid viability and cannot be functionally assessed using standard genetic approaches such as gene deletion. To facilitate genetic analysis of essential genes, we and others have assembled collections of yeast strains expressing temperature-sensitive (ts) alleles of essential genes. To explore the phenotypes caused by essential gene mutation we used a panel of genetically engineered fluorescent markers to explore the morphology of cells in the ts strain collection using high-throughput microscopy. Here, we describe the design and implementation of an online database, PhenoM (Phenomics of yeast Mutants), for storing, retrieving, visualizing and data mining the quantitative single-cell measurements extracted from micrographs of the ts mutant cells. PhenoM allows users to rapidly search and retrieve raw images and their quantified morphological data for genes of interest. The database also provides several data-mining tools, including a PhenoBlast module for phenotypic comparison between mutant strains and a Gene Ontology module for functional enrichment analysis of gene sets showing similar morphological alterations. The current PhenoM version 1.0 contains 78?194 morphological images and 1?909?914 cells covering six subcellular compartments or structures for 775 ts alleles spanning 491 essential genes. PhenoM is freely available at http://phenom.ccbr.utoronto.ca/ - Chem Biol. 2011 Sep 23;18(9):1167-78.
Activators of cylindrical proteases as antimicrobials: identification and development of small molecule activators of ClpP protease.
Leung E, Datti A, Cossette M, Goodreid J, McCaw SE, Mah M, Nakhamchik A, Ogata K, El Bakkouri M, Cheng YQ, Wodak SJ, Eger BT, Pai EF, Liu J, Gray-Owen S, Batey RA, Houry WA.
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
ClpP is a cylindrical serine protease whose ability to degrade proteins is regulated by the unfoldase ATP-dependent chaperones. ClpP on its own can only degrade small peptides. Here, we used ClpP as a target in a high-throughput screen for compounds, which activate the protease and allow it to degrade larger proteins, hence, abolishing the specificity arising from the ATP-dependent chaperones. Our screen resulted in five distinct compounds, which we designate as Activators of Self-Compartmentalizing Proteases 1 to 5 (ACP1 to 5). The compounds are found to stabilize the ClpP double-ring structure. The ACP1 chemical structure was considered to have drug-like characteristics and was further optimized to give analogs with bactericidal activity. Hence, the ACPs represent classes of compounds that can activate ClpP and that can be developed as potential novel antibiotics. - Mol Cancer Ther. 2011 Nov;10(11):2083-93. Epub 2011 Aug 22.
Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks.
Prassas I, Karagiannis GS, Batruch I, Dimitromanolakis A, Datti A, Diamandis EP.
Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada.
Cardiac glycosides (e.g., digoxin, digitoxin) constitute a diverse family of plant-derived sodium pump inhibitors that have been in clinical use for the treatment of heart-related diseases (congestive heart failure, atrial arrhythmia) for many years. Recently though, accumulating in vitro and in vivo evidence highlight potential anticancer properties of these compounds. Despite the fact that members of this family have advanced to clinical trial testing in cancer therapeutics, their cytotoxic mechanism is not yet elucidated. In this study, we investigated the cytotoxic properties of cardiac glycosides against a panel of pancreatic cancer cell lines, explored their apoptotic mechanism, and characterized the kinetics of cell death induced by these drugs. Furthermore, we deployed a high-throughput kinome screening approach and identified several kinases of the Na-K-ATPase-mediated signal transduction circuitry (epidermal growth factor receptor, Src, pkC, and mitogen-activated protein kinases) as important mediators downstream of cardiac glycoside cytotoxic action. To further extend our knowledge on their mode of action, we used mass-spectrometry-based quantitative proteomics (stable isotope labeling of amino acids in cell culture) coupled with bioinformatics to capture large-scale protein perturbations induced by a physiological dose of digitoxin in BxPC-3 pancreatic cancer cells and identified members of the interferon family as key regulators of the main protein/protein interactions downstream of digitoxin action. Hence, our findings provide more in-depth information regarding the molecular mechanisms underlying cardiac glycoside-induced cytotoxicity. - Bioorg Med Chem Lett. 2011 Sep 15;21(18):5605-9. Epub 2011 Jun 30.
Identification of a non-phosphorylated, cell permeable, small molecule ligand for the Stat3 SH2 domain.
Page BD, Fletcher S, Yue P, Li Z, Zhang X, Sharmeen S, Datti A, Wrana JL, Trudel S, Schimmer AD, Turkson J, Gunning PT.
Department of Chemistry, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON, Canada L5L 1C6.
Signal transducer and activator of transcription 3 (Stat3) protein is a cytosolic transcription factor that is aberrantly activated in numerous human cancers. Inhibitors of activated Stat3-Stat3 protein complexes have been shown to hold therapeutic promise for the treatment of human cancers harboring activated Stat3. Herein, we report the design and synthesis of a focused library of salicylic acid containing Stat3 SH2 domain binders. The most potent inhibitor, 17o, effectively disrupted Stat3-phosphopeptide complexes (K(i)=13 µM), inhibited Stat3-Stat3 protein interactions (IC(50)=19 µM) and silenced intracellular Stat3 phosphorylation and Stat3-target gene expression profiles. Inhibition of Stat3 function in both breast and multiple myeloma (MM) tumor cells correlated with induced cell death (EC(50)=10 and 16 µM, respectively). - Nat Biotechnol. 2011 Apr;29(4):361-7. Epub 2011 Mar 27.
Systematic exploration of essential yeast gene function with temperature-sensitive mutants.
Li Z, Vizeacoumar FJ, Bahr S, Li J, Warringer J, Vizeacoumar FS, Min R, Vandersluis B, Bellay J, Devit M, Fleming JA, Stephens A, Haase J, Lin ZY, Baryshnikova A, Lu H, Yan Z, Jin K, Barker S, Datti A, Giaever G, Nislow C, Bulawa C, Myers CL, Costanzo M, Gingras AC, Zhang Z, Blomberg A, Bloom K, Andrews B, Boone C.
Banting and Best Department of Medical Research and Department of Molecular Genetics, The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.
Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes. - Oncotarget. 2011 Mar;2(3):186-96.
Inhibition of SREBP1 sensitizes cells to death ligands.
Eberhard Y, Gronda M, Hurren R, Datti A, Maclean N, Ketela T, Moffat J, Wrana JL, Schimmer AD.
Princess Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada.
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. - Cancer Res. 2011 Feb 15;71(4):1385-95. Epub 2011 Feb 8.
Small molecule kinase inhibitor screen identifies polo-like kinase 1 as a target for neuroblastoma tumor-initiating cells.
Grinshtein N, Datti A, Fujitani M, Uehling D, Prakesch M, Isaac M, Irwin MS, Wrana JL, Al-Awar R, Kaplan DR.
Authors' Affiliations: Cell Biology Program and James Birrell Laboratories, The Hospital for Sick Children; Sinai-McLaughlin Assay and Robotic Technologies Facility, Samuel Lunenfeld Research Institute; Medicinal Chemistry Platform, Ontario Institute for Cancer Research; and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; and Department of Experimental Medicine and Biomedical Sciences, University of Perugia, Perugia, Italy.
Neuroblastoma (NB) is an often fatal pediatric tumor of neural crest origin. We previously isolated NB tumor-initiating cells (NB TIC) from bone marrow metastases that resemble cancer stem cells and form metastatic NB in immunodeficient animals with as few as ten cells. To identify signaling pathways important for the survival and self-renewal of NB TICs and potential therapeutic targets, we screened a small molecule library of 143 protein kinase inhibitors, including 33 in clinical trials. Cytostatic or cytotoxic drugs were identified that targeted PI3K (phosphoinositide 3-kinase)/Akt, PKC (protein kinase C), Aurora, ErbB2, Trk, and Polo-like kinase 1 (PLK1). Treatment with PLK1 siRNA or low nanomolar concentrations of BI 2536 or BI 6727, PLK1 inhibitors in clinical trials for adult malignancies, were cytotoxic to TICs whereas only micromolar concentrations of the inhibitors were cytotoxic for normal pediatric neural stem cells. Furthermore, BI 2536 significantly inhibited TIC tumor growth in a therapeutic xenograft model, both as a single agent and in combination with irinotecan, an active agent for relapsed NB. Our findings identify candidate kinases that regulate TIC growth and survival and suggest that PLK1 inhibitors are an attractive candidate therapy for metastatic NB. Cancer Res; 71(4); 1385-95. ©2011 AACR. - Sci Transl Med. 2011 Jan 26;3(67):67ra7
Uroporphyrinogen decarboxylase is a radiosensitizing target for head and neck cancer.
Ito E, Yue S, Moriyama EH, Hui AB, Kim I, Shi W, Alajez NM, Bhogal N, Li G, Datti A, Schimmer AD, Wilson BC, Liu PP, Durocher D, Neel BG, O'Sullivan B, Cummings B, Bristow R, Wrana J, Liu FF.
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5G 2M9.
Head and neck cancer (HNC) is the eighth most common malignancy worldwide, comprising a diverse group of cancers affecting the head and neck region. Despite advances in therapeutic options over the last few decades, treatment toxicities and overall clinical outcomes have remained disappointing, thereby underscoring a need to develop novel therapeutic approaches in HNC treatment. Uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified from an RNA interference-based high-throughput screen as a tumor-selective radiosensitizing target for HNC. UROD knockdown plus radiation induced caspase-mediated apoptosis and cell cycle arrest in HNC cells in vitro and suppressed the in vivo tumor-forming capacity of HNC cells, as well as delayed the growth of established tumor xenografts in mice. This radiosensitization appeared to be mediated by alterations in iron homeostasis and increased production of reactive oxygen species, resulting in enhanced tumor oxidative stress. Moreover, UROD was significantly overexpressed in HNC patient biopsies. Lower preradiation UROD mRNA expression correlated with improved disease-free survival, suggesting that UROD could potentially be used to predict radiation response. UROD down-regulation also radiosensitized several different models of human cancer, as well as sensitized tumors to chemotherapeutic agents, including 5-fluorouracil, cisplatin, and paclitaxel. Thus, our study has revealed UROD as a potent tumor-selective sensitizer for both radiation and chemotherapy, with potential relevance to many human malignancies. - Blood. 2011 Feb 10;117(6):1986-97. Epub 2010 Dec 6.
A small-molecule inhibitor of D-cyclin transactivation displays preclinical efficacy in myeloma and leukemia via phosphoinositide 3-kinase pathway.
Mao X, Cao B, Wood TE, Hurren R, Tong J, Wang X, Wang W, Li J, Jin Y, Sun W, Spagnuolo PA, MacLean N, Moran MF, Datti A, Wrana J, Batey RA, Schimmer AD.
Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou, China.
D-cyclins are universally dysregulated in multiple myeloma and frequently overexpressed in leukemia. To better understand the role and impact of dysregulated D-cyclins in hematologic malignancies, we conducted a high-throughput screen for inhibitors of cyclin D2 transactivation and identified 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161), which inhibited the expression of cyclins D1, D2, and D3 and arrested cells at the G(0)/G(1) phase. After D-cyclin suppression, S14161 induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of leukemia, S14161 inhibited tumor growth without evidence of weight loss or gross organ toxicity. Mechanistically, S14161 inhibited the activity of phosphoinositide 3-kinase in intact cells and the activity of the phosphoinositide 3-kinases a, ß, d, and ? in a cell-free enzymatic assay. In contrast, it did not inhibit the enzymatic activities of other related kinases, including the mammalian target of rapamycin, the DNA-dependent protein kinase catalytic subunit, and phosphoinositide-dependent kinase-1. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation via the phosphoinositide 3-kinase/protein kinase B signaling pathway. Given its potent antileukemia and antimyeloma activity and minimal toxicity, S14161 could be developed as a novel agent for blood cancer therapy. - Mol Cell. 2010 Nov 24;40(4):619-31. Epub 2010 Nov 4.
The MMS22L-TONSL complex mediates recovery from replication stress and homologous recombination.
O'Donnell L, Panier S, Wildenhain J, Tkach JM, Al-Hakim A, Landry MC, Escribano-Diaz C, Szilard RK, Young JT, Munro M, Canny MD, Kolas NK, Zhang W, Harding SM, Ylanko J, Mendez M, Mullin M, Sun T, Habermann B, Datti A, Bristow RG, Gingras AC, Tyers MD, Brown GW, Durocher D.
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks. - J Biol Chem. 2010 Nov 26;285(48):37476-90. Epub 2010 Sep 13.
Inhibition of osteoclast bone resorption by disrupting vacuolar H+-ATPase a3-B2 subunit interaction.
Kartner N, Yao Y, Li K, Crasto GJ, Datti A, Manolson MF.
Dental Research Institute, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6 Canada.
Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-?B ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of ~1.2 µm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-?B ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis. - EMBO Mol Med. 2010 Sep;2(9):371-84.
Selective targeting of neuroblastoma tumour-initiating cells by compounds identified in stem cell-based small molecule screens.
Smith KM, Datti A, Fujitani M, Grinshtein N, Zhang L, Morozova O, Blakely KM, Rotenberg SA, Hansford LM, Miller FD, Yeger H, Irwin MS, Moffat J, Marra MA, Baruchel S, Wrana JL, Kaplan DR.
Cell Biology Program and James Burrell Laboratories, The Hospital for Sick Children, Toronto, Canada.
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. - Blood. 2010 Nov 4;116(18):3593-603. Epub 2010 Jul 19.
The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells.
Sharmeen S, Skrtic M, Sukhai MA, Hurren R, Gronda M, Wang X, Fonseca SB, Sun H, Wood TE, Ward R, Minden MD, Batey RA, Datti A, Wrana J, Kelley SO, Schimmer AD.
Princess Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada.
To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia. - Cell Stem Cell. 2010 Jul 2;7(1):64-77. Epub 2010 Jun 17.
Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming.
Samavarchi-Tehrani P, Golipour A, David L, Sung HK, Beyer TA, Datti A, Woltjen K, Nagy A, Wrana JL.
Center for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. PAPERCLIP: - J Cell Biol. 2010 Jan 11;188(1):69-81.
Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis
Vizeacoumar FJ, van Dyk N, Vizeacoumar FS, Cheung V, Li J, Sydorskyy Y, Case N, Li Z, Datti A, Nislow C, Raught B, Zhang Z, Frey B, Bloom K, Boone C, Andrews BJ.
Banting & Best Department of Medical Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada
We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. - Mol Syst Biol. 2009;5:315. Epub 2009 Oct 13
Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway.
Miller BW, Lau G, Grouios C, Mollica E, Barrios-Rodiles M, Liu Y, Datti A, Morris Q, Wrana JL, Attisano L.
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
Large-scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi-dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein-protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA-mediated gene knockdown on Wnt signaling. Meta-analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates beta-catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators. - Mol Pharmacol. 2009 Nov;76(5):969-83. Epub 2009 Aug 4.
Potential use of cetrimonium bromide as an apoptosis-promoting anticancer agent for head and neck cancer.
Ito E, Yip KW, Katz D, Fonseca SB, Hedley DW, Chow S, Xu GW, Wood TE, Bastianutto C, Schimmer AD, Kelley SO, Liu FF.
Department of Medical Biophysics, University of Toronto, Ontario, Canada.
A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with gamma radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H(+)-ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G(1) cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models. - Blood. 2009 Oct 1;114(14):3064-73. Epub 2009 Jul 9.
Chelation of intracellular iron with the anti-fungal agent ciclopirox olamine induces cell death in leukemia and myeloma cells.
Eberhard Y, McDermott S, Wang X, Gronda M, Venugopal A, Wood TE, Hurren R, Datti A, Batey RA, Wrana J, Antholine WE, Dick J, Schimmer AD
The Princess Margaret Hospital, The Ontario Cancer Institute, Toronto, ON, Canada.
Off-patent drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. To identify such compounds, we conducted two independent cell-based chemical screens and identified the antimicrobial, ciclopirox olamine (CPX) in both screens. CPX decreased cell growth and viability of malignant leukemia, myeloma, and solid tumor cell lines as well as primary AML patient samples at low micromolar concentrations that appear pharmacologically achievable. Furthermore, oral CPX decreased tumor weight and volume in 3 mouse models of leukemia by up to 65% compared to control without evidence of weight loss or gross organ toxicity. In addition, oral CPX prevented the engraftment of primary AML cells in NOD/SCID mouse models, thereby establishing its ability to target leukemia stem cells. Mechanistically, CPX bound intracellular iron and this intracellular iron chelation was functionally important for its cytotoxicity. By electron paramagnetic resonance, CPX inhibited the iron-dependent enzyme ribonucleotide reductase at concentrations associated with cell death. Thus, in summary, CPX has previously unrecognized anti-cancer activity at concentrations that are pharmacologically achievable. Therefore, CPX could be rapidly repurposed for the treatment of malignancies including leukemia and myeloma. - Cell (2009) 136:420-34
The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage.
Stewart GS, Panier S, Townsend K, Al-Hakim AK, Kolas NK, Miller ES, Nakada S, Ylanko J, Olivarius S, Mendez M, Oldreive C, Wildenhain J, Tagliaferro A, Pelletier L, Taubenheim N, Durandy A, Byrd PJ, Stankovic T, Taylor AM, Durocher D
Cancer Research UK, Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, UK.
The biological response to DNA double-strand breaks acts to preserve genome integrity. Individuals bearing inactivating mutations in components of this response exhibit clinical symptoms that include cellular radiosensitivity, immunodeficiency, and cancer predisposition. The archetype for such disorders is Ataxia-Telangiectasia caused by biallelic mutation in ATM, a central component of the DNA damage response. Here, we report that the ubiquitin ligase RNF168 is mutated in the RIDDLE syndrome, a recently discovered immunodeficiency and radiosensitivity disorder. We show that RNF168 is recruited to sites of DNA damage by binding to ubiquitylated histone H2A. RNF168 acts with UBC13 to amplify the RNF8-dependent histone ubiquitylation by targeting H2A-type histones and by promoting the formation of lysine 63-linked ubiquitin conjugates. These RNF168-dependent chromatin modifications orchestrate the accumulation of 53BP1 and BRCA1 to DNA lesions, and their loss is the likely cause of the cellular and developmental phenotypes associated with RIDDLE syndrome. - Nat. Methods (2009) 6:91-7
An experimentally derived confidence score for binary protein-protein interactions
Braun P, Tasan M, Dreze M, Barrios-Rodiles M, Lemmens I, Yu H, Sahalie JM, Murray RR, Roncari L, de Smet AS, Venkatesan K, Rual JF, Vandenhaute J, Cusick ME, Pawson T, Hill DE, Tavernier J, Wrana JL, Roth FP, Vidal M
Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA.
Information on protein-protein interactions is of central importance for many areas of biomedical research. At present no method exists to systematically and experimentally assess the quality of individual interactions reported in interaction mapping experiments. To provide a standardized confidence-scoring method that can be applied to tens of thousands of protein interactions, we have developed an interaction tool kit consisting of four complementary, high-throughput protein interaction assays. We benchmarked these assays against positive and random reference sets consisting of well documented pairs of interacting human proteins and randomly chosen protein pairs, respectively. A logistic regression model was trained using the data from these reference sets to combine the assay outputs and calculate the probability that any newly identified interaction pair is a true biophysical interaction once it has been tested in the tool kit. This general approach will allow a systematic and empirical assignment of confidence scores to all individual protein-protein interactions in interactome networks. - Clin.Cancer Res. (2008) 14:5778-84
High-throughput screening identifies cardiac glycosides as potent inhibitors of human tissue kallikrein expression: implications for cancer therapies.
Prassas I, Paliouras M, Datti A, Diamandis EP
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
PURPOSE: Human tissue kallikreins (KLK) comprise a subgroup of 15 homologous secreted serine proteases. Primarily known for their clinical use as cancer biomarkers (e.g., PSA), KLKs have recently been directly implicated in cancer-related processes, including invasion, angiogenesis, and tumor growth regulation. Therefore, the identification of compounds that would modulate expression of KLKs might be of considerable therapeutic value. EXPERIMENTAL DESIGN: A cell-based high-throughput screening (HTS) of three small molecule libraries ( approximately 4,500 compounds) was undertaken; KLK expression in the breast cancer cell line MDA-MB-468 was assessed with sensitive ELISAs. RESULTS: The initial screening resulted in 66 "putative hits" that decreased KLK5 expression by at least 50% over control. Secondary screening and mini-dose-response assays resulted in 21 "validated hits." These 21 compounds were clustered in only three distinct functional families and were further analyzed in vitro to determine their effectiveness (IC(50)s). Hits that failed to show dose-responsiveness or interfered with the viability of the cells were excluded. Multiple members of the cardiac glycoside family were found to be novel inhibitors of KLK expression, acting at low concentrations (10-50 nmol/L). Furthermore, members of the same family induced marked decreases in c-MYC and c-FOS expression, in a dose-dependent manner that correlated the KLK inhibition, suggesting a transcriptional mechanism of regulation of KLK expression. CONCLUSIONS: We conclude that cardiac glycosides can dramatically suppress the transcription of KLKs and that these effects may be linked to proto-oncogene (c-myc/fos) expression. These findings may partially explain the recently realized antineoplastic actions of cardiac glycosides. - Cancer Res. (2008) 68:6688-97
Inhibition of the sodium/potassium ATPase impairs N-glycan expression and function.
Beheshti Zavareh R, Lau K, Hurren R, Datti A, Gronda M., Cheung P, Liu W, Wasylishen A, Boutros P, Shi H, Ashline DJ, Penn LZ, Reinhold V, Ezzat S, Wrana J, Rose D, Schachter H, Dennis JW, Schimmer AD
Ontario Cancer Institute, Princess Margaret Hospital, Canada.
Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with beta1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA-induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA-induced agglutination and cell death. All of the CGs tested inhibited L-PHA-induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC(50) of 60 +/- 20 nmol/L. Digoxin also increased the fraction of some concanavalin A-binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc(1)Man(3)GlcNAc(2)Fuc(1) and GlcNAc(2)Man(3)GlcNAc(2)Fuc(1) oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation-mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients. - Blood (2008) 112:760-9
Cyproheptadine displays preclinical activity in myeloma and leukemia.
Mao X, Liang SB, Hurren R, Gronda M, Chow S, Xu GW, Wang X, Beheshti Zavareh R, Jamal N, Messner H, Hedley DW, Datti A, Wrana JL, Zhu Y, Shi CX, Lee K, Tiedemann R, Trudel S, Stewart AK, Schimmer AD
Princess Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada.
D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer. - Biotechniques (2008) 44:ix-xiv
Increased efficiency for performing colony formation assays in 96-well plates: applicability to combination therapies and high-throughput screening.
Katz D, Ito E, Lau KS, Mocanu JD, Bastianutto C, Schimmer AD, Liu FF
University of Toronto, Canada.
The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its ability to evaluate combination therapies was investigated by the generation of dose-response curves for the combination of cisplatin and radiation therapy on FaDu and A549 cells. The 96-well CFA was then transferred to a robotic platform for evaluating its potential as a high-throughput screening (HTS) readout. The LOPAC1280 library was screened against FaDu cells, and eight putative hits were identified. Using the 96-well CFA to validate the eight putative chemicals, six of the eight were confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and combination therapies in vitro, providing a possible readout that could be used on a HTS platform. - Apoptosis (2008) 13:413-22
A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization.
Xu GW, Mawji IA, Macrae CJ, Koch CA, Datti A, Wrana JL, Dennis JW, Schimmer AD
Ontario Cancer Institute, Princess Margaret Hospital, 610 University Ave, M5G 2M9, Toronto, ON, Canada.
p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity. - Science (2007) 318:1637-40
Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.
Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, Durocher D
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G1X5, Ontario, Canada.
Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination. - Blood (2007) 110:4047-54
A chemical biology screen identifies glucocorticoids that regulate c-maf expression by increasing its proteasomal degradation through up-regulation of ubiquitin.
Mao X, Stewart AK, Hurren R, Datti A, Zhu X, Zhu Y, Shi C, Lee K, Tiedemann R, Eberhard Y, Trudel S, Liang S, Corey SJ, Gillis LC, Barber DL, Wrana JL, Ezzat S, Schimmer AD
Princess Margaret Hospital, Ontario Cancer Institute, Toronto, ON, Canada.
The oncogene c-maf is frequently overexpressed in multiple myeloma cell lines and patient samples and contributes to increased cellular proliferation in part by inducing cyclin D2 expression. To identify regulators of c-maf, we developed a chemical screen in NIH3T3 cells stably overexpressing c-maf and the cyclin D2 promoter driving luciferase. From a screen of 2400 off-patent drugs and chemicals, we identified glucocorticoids as c-maf-dependent inhibitors of cyclin D2 transactivation. In multiple myeloma cell lines, glucocorticoids reduced levels of c-maf protein without influencing corresponding mRNA levels. Subsequent studies demonstrated that glucocorticoids increased ubiquitination-dependent degradation of c-maf and up-regulated ubiquitin C mRNA. Moreover, ectopic expression of ubiquitin C recapitulated the effects of glucocorticoids, demonstrating regulation of c-maf protein through the abundance of the ubiquitin substrate. Thus, using a chemical biology approach, we identified a novel mechanism of action of glucocorticoids and a novel mechanism by which levels of c-maf protein are regulated by the abundance of the ubiquitin substrate. - Cancer Res. (2007) 67:9771-80
Complex N-glycan and metabolic control in tumor cells.
Mendelsohn R, Cheung P, Berger L, Partridge E, Lau K, Datti A, Pawling J, Dennis JW
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada.
Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5) produces beta1,6GlcNAc-branched complex N-glycans on cell surface glycoproteins that bind to galectins and promote surface residency of glycoproteins, including cytokine receptors. Carcinoma cells from polyomavirus middle T (PyMT) transgenic mice on a Mgat5-/- background have reduced surface levels of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) receptors and are less sensitive to acute stimulation by cytokines in vitro compared with PyMT Mgat5+/+ tumor cells but are nonetheless tumorigenic when injected into mice. Here, we report that PyMT Mgat5-/- cells are reduced in size, checkpoint impaired, and following serum withdrawal, fail to down-regulate glucose transport, protein synthesis, reactive oxygen species (ROS), and activation of Akt and extracellular signal-regulated kinase. To further characterize Mgat5+/+ and Mgat5-/- tumor cells, a screen of pharmacologically active compounds was done. Mgat5-/- tumor cells were comparatively hypersensitive to the ROS inducer 2,3-dimethoxy-1,4-naphthoquinone, hyposensitive to tyrosine kinase inhibitors, to Golgi disruption by brefeldin A, and to mitotic arrest by colcemid, hydroxyurea, and camptothecin. Finally, regulation of ROS, glucose uptake, and sensitivities to EGF and TGF-beta were rescued by Mgat5 expression or by hexosamine supplementation to complex N-glycan biosynthesis in Mgat5-/- cells. Our results suggest that complex N-glycans sensitize tumor cells to growth factors, and Mgat5 is required to balance responsiveness to growth and arrest cues downstream of metabolic flux. - Cancer Res. (2007) 67:8307-15
A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis
Mawji IA, Simpson CD, Gronda M, Williams MA, Hurren R, Henderson CJ, Datti A, Wrana JL, Schimmer AD
Ontario Cancer Institute, Princess Margaret Hospital, Mt Sinai Hospital, Toronto, Ontario, Canada.
Malignant epithelial cells with metastatic potential resist apoptosis that normally occurs upon loss of anchorage from the extracellular matrix, a process termed "anoikis." Resistance to anoikis enables malignant cells to survive in an anchorage-independent manner, which leads to the formation of distant metastases. To understand the regulation of anoikis, we designed, automated, and conducted a high-throughput chemical screen for anoikis sensitizers. PPC-1 anoikis-resistant prostate cancer cells were seeded in hydrogel-coated ultralow binding plates for suspension conditions and standard tissue culture plates to promote adhesion. After seeding, cells were treated with aliquots from a library of previously characterized small molecules, and viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, assay. From this chemical screen, we identified anisomycin that induced apoptosis in suspension conditions, but was not toxic to these cells grown under adherent conditions. Anisomycin sensitized cells to anoikis by decreasing levels of the caspase-8 inhibitor FLIP and subsequently activating the death receptor pathway of caspase activation. Although anisomycin activated c-Jun-NH(2)-kinase and p38, these kinases were not functionally important for the effect of anisomycin on anoikis and FLIP. Rather, anisomycin decreased FLIP and sensitized cells to anoikis by inhibiting its protein synthesis. Finally, we showed that anisomycin decreased distal tumor formation in a mouse model of prostate cancer metastases. Thus, a novel chemical screen identified anisomycin as an anoikis sensitizer that acts by decreasing FLIP protein synthesis. Our results suggest that FLIP is a suppressor of anoikis and inhibiting FLIP protein synthesis may be a useful antimetastatic strategy. - Clin.Cancer Res. (2006) 12:5557-69
Benzethonium chloride: a novel anticancer agent identified by using a cell-based small-molecule screen.
Yip KW, Mao X, Au PY, Hedley DW, Chow S, Dalili S, Mocanu JD, Bastianutto C, Schimmer A, Liu FF
Department of Medical Biophysics, University of Toronto, and Department of Medical Oncology, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada.
PURPOSE: This study aims to identify a novel therapeutic agent for head and neck cancer and to evaluate its antitumor efficacy. EXPERIMENTAL DESIGN: A cell-based and phenotype-driven high-throughput screening of approximately 2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal cancer) and GM05757 (primary normal human fibroblast) lines. The "hit" compound was assayed for efficacy in combination with standard therapeutics on a panel of human cancer cell lines. Furthermore, its mode of action (using transmission electron microscopy and flow cytometry) and its in vivo efficacy (using xenograft models) were evaluated. RESULTS: Benzethonium chloride was identified as a novel cancer-specific compound. For benzethonium (48-hour incubation), the dose required to reduce cell viability by 50% was 3.8 micromol/L in FaDu, 42.2 micromol/L in NIH 3T3, 5.3 micromol/L in C666-1, and 17.0 micromol/L in GM05757. In vitro, this compound did not interfere with the effects of cisplatin, 5-fluorouracil, or gamma-irradiation. Benzethonium chloride induced apoptosis and activated caspases after 12 hours. Loss of mitochondrial membrane potential (DeltaPsiM) preceded cytosolic Ca2+ increase and cell death. In vivo, benzethonium chloride ablated the tumor-forming ability of FaDu cells, delayed the growth of xenograft tumors, and combined additively with local tumor radiation therapy. Evaluation of benzethonium chloride on the National Cancer Institute/NIH Developmental Therapeutics Program 60 human cancer cell lines revealed broad-range antitumor activity. CONCLUSIONS: This high-throughput screening identified a novel antimicrobial compound with significant broad-spectrum anticancer activity. - Mol Cancer Ther. (2006) 5:2234-40
Potential use of alexidine dihydrochloride as an apoptosis-promoting anticancer agent.
Yip KW, Ito E, Mao X, Au PY, Hedley DW, Mocanu JD, Bastianutto C, Schimmer A, Liu FF
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Despite advances in surgery, radiation, and chemotherapy, novel therapeutics are needed for head and neck cancer treatment. The objective of this current study was to evaluate alexidine dihydrochloride as a novel compound lead for head and neck cancers. Using a tetrazolium-based assay, the dose required to reduce cell viability by 50% (ED50) was found to be approximately 1.8 micromol/L in FaDu (human hypopharyngeal squamous cancer) and approximately 2.6 micromol/L in C666-1 (human undifferentiated nasopharyngeal cancer) cells. In contrast, the ED50 values were much higher in untransformed cells, specifically at approximately 8.8 micromol/L in GM05757 (primary normal human fibroblast), approximately 8.9 micromol/L in HNEpC (primary normal human nasal epithelial), and approximately 19.6 micromol/L in NIH/3T3 (mouse embryonic fibroblast) cells. Alexidine dihydrochloride did not interfere with the activities of cisplatin, 5-fluorouracil, or radiation, and interacted in a less-than-additive manner. DNA content analyses and Hoechst 33342 staining revealed that this compound induced apoptosis. Alexidine dihydrochloride-induced mitochondrial damage was visualized using transmission electron microscopy. Mitochondrial membrane potential (DeltaPsiM) depolarization was detectable after only 3 hours of treatment, and was followed by cytosolic Ca2+ increase along with loss of membrane integrity/cell death. Caspase-2 and caspase-9 activities were detectable at 12 hours, caspase-8 at 24 hours, and caspase-3 at 48 hours. FaDu cell clonogenic survival was reduced to < 5% with 1 micromol/L alexidine dihydrochloride, and, correspondingly, this compound decreased the in vivo tumor-forming potential of FaDu cells. Thus, we have identified alexidine dihydrochloride as the first bisbiguanide compound with anticancer specificity. - Cancer Res. 2006 Apr 1;66(7):3558-66
Chemical enhancers of cytokine signaling that suppress microfilament turnover and tumor cell growth.
Park HJ, Partridge E, Cheung P, Pawling J, Donovan R, Wrana JL, Dennis JW
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue R988, Toronto, Ontario, Canada M5G 1X5.
The transforming growth factor-beta (TGF-beta) family of cytokines regulates cell proliferation, morphogenesis, and specialized cell functions in metazoans. Herein, we screened a compound library for modifiers of TGF-beta signaling in NMuMG epithelial cells using a cell-based assay to measure Smad2/3 nuclear translocation. We identified five enhancers of TGF-beta signaling that share a core structure of diethyl 2-(anilinomethylene)malonate (DAM), and D(50) values of 1 to 4 micromol/L. Taking advantage of the Mgat5 mutant phenotype of accelerated receptor loss to endocytosis, we determined that DAM-1976 restored the sensitivity of Mgat5(-/-) carcinoma cells to both TGF-beta and epidermal growth factor (EGF). In Mgat5 mutant and wild-type carcinoma cells, DAM-1976 enhanced and prolonged TGF-beta- and EGF-dependent Smad2/3 and Erk activation, respectively. DAM-1976 reduced ligand-dependent EGF receptor endocytosis, actin microfilament turnover, and cell spreading, suggesting that the compound attenuates vesicular trafficking. Hyperactivation of intracellular signaling has the potential to suppress tumor cell growth and, in this regard, DAM-1976 represents a new pharmacophore that increases basal activation of Smad2/3 and Erk, inhibits microfilament remodeling, and suppresses carcinoma cell growth. - Science. 2005 Mar 11;307(5715):1621-5
High-throughput mapping of a dynamic signaling network in mammalian cells.
Barrios-Rodiles M, Brown KR, Ozdamar B, Bose R, Liu Z, Donovan RS, Shinjo F, Liu Y, Dembowy J, Taylor IW, Luga V, Przulj N, Robinson M, Suzuki H, Hayashizaki Y, Jurisica I, Wrana JL
Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5.
Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.