Publications by authors named "Troy Ketela"

42 Publications

Irx3 and Irx5 in Ins2-Cre cells regulate hypothalamic postnatal neurogenesis and leptin response.

Nat Metab 2021 05 15;3(5):701-713. Epub 2021 Apr 15.

Program in Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada.

Obesity is mainly due to excessive food intake. IRX3 and IRX5 have been suggested as determinants of obesity in connection with the intronic variants of FTO, but how these genes contribute to obesity via changes in food intake remains unclear. Here, we show that mice doubly heterozygous for Irx3 and Irx5 mutations exhibit lower food intake with enhanced hypothalamic leptin response. By lineage tracing and single-cell RNA sequencing using the Ins2-Cre system, we identify a previously unreported radial glia-like neural stem cell population with high Irx3 and Irx5 expression in early postnatal hypothalamus and demonstrate that reduced dosage of Irx3 and Irx5 promotes neurogenesis in postnatal hypothalamus leading to elevated numbers of leptin-sensing arcuate neurons. Furthermore, we find that mice with deletion of Irx3 in these cells also exhibit a similar food intake and hypothalamic phenotype. Our results illustrate that Irx3 and Irx5 play a regulatory role in hypothalamic postnatal neurogenesis and leptin response.
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http://dx.doi.org/10.1038/s42255-021-00382-yDOI Listing
May 2021

A genome-wide CRISPR/Cas9 screen in acute myeloid leukemia cells identifies regulators of TAK-243 sensitivity.

JCI Insight 2021 03 8;6(5). Epub 2021 Mar 8.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

TAK-243 is a first-in-class inhibitor of ubiquitin-like modifier activating enzyme 1 that catalyzes ubiquitin activation, the first step in the ubiquitylation cascade. Based on its preclinical efficacy and tolerability, TAK-243 has been advanced to phase I clinical trials in advanced malignancies. Nonetheless, the determinants of TAK-243 sensitivity remain largely unknown. Here, we conducted a genome-wide CRISPR/Cas9 knockout screen in acute myeloid leukemia (AML) cells in the presence of TAK-243 to identify genes essential for TAK-243 action. We identified BEN domain-containing protein 3 (BEND3), a transcriptional repressor and a regulator of chromatin organization, as the top gene whose knockout confers resistance to TAK-243 in vitro and in vivo. Knockout of BEND3 dampened TAK-243 effects on ubiquitylation, proteotoxic stress, and DNA damage response. BEND3 knockout upregulated the ATP-binding cassette efflux transporter breast cancer resistance protein (BCRP; ABCG2) and reduced the intracellular levelsof TAK-243. TAK-243 sensitivity correlated with BCRP expression in cancer cell lines of different origins. Moreover, chemical inhibition and genetic knockdown of BCRP sensitized intrinsically resistant high-BCRP cells to TAK-243. Thus, our data demonstrate that BEND3 regulates the expression of BCRP for which TAK-243 is a substrate. Moreover, BCRP expression could serve as a predictor of TAK-243 sensitivity.
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http://dx.doi.org/10.1172/jci.insight.141518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021101PMC
March 2021

Functional genomics identifies new synergistic therapies for retinoblastoma.

Oncogene 2020 07 22;39(31):5338-5357. Epub 2020 Jun 22.

Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health, Toronto, ON, Canada.

Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1 and RB1;MYCN orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53-BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53-p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53-BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.
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http://dx.doi.org/10.1038/s41388-020-1372-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391301PMC
July 2020

Mitochondrial carrier homolog 2 is necessary for AML survival.

Blood 2020 07;136(1):81-92

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.
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http://dx.doi.org/10.1182/blood.2019000106DOI Listing
July 2020

Inhibition of mitochondrial translation overcomes venetoclax resistance in AML through activation of the integrated stress response.

Sci Transl Med 2019 10;11(516)

Princess Margaret Cancer Centre, Toronto, Ontario M5G 1L7, Canada.

Venetoclax is a specific B cell lymphoma 2 (BCL-2) inhibitor with promising activity against acute myeloid leukemia (AML), but its clinical efficacy as a single agent or in combination with hypomethylating agents (HMAs), such as azacitidine, is hampered by intrinsic and acquired resistance. Here, we performed a genome-wide CRISPR knockout screen and found that inactivation of genes involved in mitochondrial translation restored sensitivity to venetoclax in resistant AML cells. Pharmacologic inhibition of mitochondrial protein synthesis with antibiotics that target the ribosome, including tedizolid and doxycycline, effectively overcame venetoclax resistance. Mechanistic studies showed that both tedizolid and venetoclax suppressed mitochondrial respiration, with the latter demonstrating inhibitory activity against complex I [nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase] of the electron transport chain (ETC). The drugs cooperated to activate a heightened integrated stress response (ISR), which, in turn, suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. Combination treatment with tedizolid and venetoclax was superior to either agent alone in reducing leukemic burden in mice engrafted with treatment-resistant human AML. The addition of tedizolid to azacitidine and venetoclax further enhanced the killing of resistant AML cells in vitro and in vivo. Our findings demonstrate that inhibition of mitochondrial translation is an effective approach to overcoming venetoclax resistance and provide a rationale for combining tedizolid, azacitidine, and venetoclax as a triplet therapy for AML.
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http://dx.doi.org/10.1126/scitranslmed.aax2863DOI Listing
October 2019

Evaluation of methods to assign cell type labels to cell clusters from single-cell RNA-sequencing data.

F1000Res 2019 15;8. Epub 2019 Mar 15.

Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, 94143, USA.

Identification of cell type subpopulations from complex cell mixtures using single-cell RNA-sequencing (scRNA-seq) data includes automated steps from normalization to cell clustering. However, assigning cell type labels to cell clusters is often conducted manually, resulting in limited documentation, low reproducibility and uncontrolled vocabularies. This is partially due to the scarcity of reference cell type signatures and because some methods support limited cell type signatures. In this study, we benchmarked five methods representing first-generation enrichment analysis (ORA), second-generation approaches (GSEA and GSVA), machine learning tools (CIBERSORT) and network-based neighbor voting (METANEIGHBOR), for the task of assigning cell type labels to cell clusters from scRNA-seq data. We used five scRNA-seq datasets: human liver, 11 Tabula Muris mouse tissues, two human peripheral blood mononuclear cell datasets, and mouse retinal neurons, for which reference cell type signatures were available. The datasets span Drop-seq, 10X Chromium and Seq-Well technologies and range in size from ~3,700 to ~68,000 cells. Our results show that, in general, all five methods perform well in the task as evaluated by receiver operating characteristic curve analysis (average area under the curve (AUC) = 0.91, sd = 0.06), whereas precision-recall analyses show a wide variation depending on the method and dataset (average AUC = 0.53, sd = 0.24). We observed an influence of the number of genes in cell type signatures on performance, with smaller signatures leading more frequently to incorrect results. GSVA was the overall top performer and was more robust in cell type signature subsampling simulations, although different methods performed well using different datasets. METANEIGHBOR and GSVA were the fastest methods. CIBERSORT and METANEIGHBOR were more influenced than the other methods by analyses including only expected cell types. We provide an extensible framework that can be used to evaluate other methods and datasets at https://github.com/jdime/scRNAseq_cell_cluster_labeling.
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http://dx.doi.org/10.12688/f1000research.18490.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720041PMC
June 2020

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Am J Physiol Renal Physiol 2019 12 30;317(6):F1593-F1604. Epub 2019 Sep 30.

Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for , , or , which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.
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http://dx.doi.org/10.1152/ajprenal.00195.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962514PMC
December 2019

Functional CRISPR and shRNA Screens Identify Involvement of Mitochondrial Electron Transport in the Activation of Evofosfamide.

Mol Pharmacol 2019 06 12;95(6):638-651. Epub 2019 Apr 12.

Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health Sciences (F.W.H., C.R.H., A.K., I.S., W.R.W.), Maurice Wilkins Centre for Molecular Biodiscovery (F.W.H., A.J.R.H., C.G.P., W.R.W.), School of Biological Sciences, Faculty of Science (J.B.L.D., A.J.R.H.), and Department of Molecular Medicine and Pathology, School of Medical Sciences, Faculty of Medical and Health Sciences (P.T., P.M.K., C.G.P., S.K.B.), University of Auckland, Auckland, New Zealand; Threshold Pharmaceuticals, South San Francisco, California (F.M., C.P.H.); Princess Margaret Genomics Centre (T.W.K.) and Princess Margaret Cancer Centre (S.M., Z.S., B.G.W.), University Health Network, and Departments of Radiation Oncology (B.G.W.) and Medical Biophysics (B.G.W.), University of Toronto, Toronto, Ontario, Canada.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ( zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors , (), and , in addition to the key regulator of mitochondrial function, , and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes , , and and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.
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http://dx.doi.org/10.1124/mol.118.115196DOI Listing
June 2019

The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

Cell Stem Cell 2019 04 28;24(4):621-636.e16. Epub 2019 Mar 28.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.
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http://dx.doi.org/10.1016/j.stem.2019.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137093PMC
April 2019

The Inhibitory NKR-P1B:Clr-b Recognition Axis Facilitates Detection of Oncogenic Transformation and Cancer Immunosurveillance.

Cancer Res 2018 07 24;78(13):3589-3603. Epub 2018 Apr 24.

Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

Natural killer (NK) cells express receptors specific for MHC class I (MHC-I) molecules involved in "missing-self" recognition of cancer and virus-infected cells. Here we elucidate the role of MHC-I-independent NKR-P1B:Clr-b interactions in the detection of oncogenic transformation by NK cells. Ras oncogene overexpression was found to promote a real-time loss of Clr-b on mouse fibroblasts and leukemia cells, mediated in part via the Raf/MEK/ERK and PI3K pathways. Ras-driven Clr-b downregulation occurred at the level of the () promoter, nascent Clr-b transcripts, and cell surface Clr-b protein, in turn promoting missing-self recognition via the NKR-P1B inhibitory receptor. Both Ras- and c-Myc-mediated Clr-b loss selectively augmented cytotoxicity of oncogene-transformed leukemia cells by NKR-P1B NK cells and enhanced rejection by WT mice Interestingly, genetic ablation of either one (Clr-b) or two Clr-b alleles (Clr-b) enhanced survival of Eμ-cMyc transgenic mice in a primary lymphoma model despite preferential rejection of Clr-b hematopoietic cells previously observed following adoptive transfer into naïve wild-type mice Collectively, these findings suggest that the inhibitory NKR-P1B:Clr-b axis plays a beneficial role in innate detection of oncogenic transformation via NK-cell-mediated cancer immune surveillance, in addition to a pathologic role in the immune escape of primary lymphoma cells in Eμ-cMyc mice These results provide a model for the human NKR-P1A:LLT1 system in cancer immunosurveillance in patients with lymphoma and suggest it may represent a target for immune checkpoint therapy. A mouse model shows that an MHC-independent NK-cell recognition axis enables the detection of leukemia cells, with implications for a novel immune checkpoint therapy target in human lymphoma. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-1688DOI Listing
July 2018

Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling.

Cell Rep 2017 12;21(13):3970-3986

Program in Neuroscience and Mental Health, Hospital for Sick Children, Toronto, ON M5G 1L7, Canada; Institute of Medical Science , University of Toronto, Toronto, ON M5G 1A8, Canada; Department of Molecular Genetics , University of Toronto, Toronto, ON M5G 1A8, Canada; Department of Physiology, University of Toronto, Toronto, ON M5G 1A8, Canada. Electronic address:

Adult neural stem cells (NSCs) derive from embryonic precursors, but little is known about how or when this occurs. We have addressed this issue using single-cell RNA sequencing at multiple developmental time points to analyze the embryonic murine cortex, one source of adult forebrain NSCs. We computationally identify all major cortical cell types, including the embryonic radial precursors (RPs) that generate adult NSCs. We define the initial emergence of RPs from neuroepithelial stem cells at E11.5. We show that, by E13.5, RPs express a transcriptional identity that is maintained and reinforced throughout their transition to a non-proliferative state between E15.5 and E17.5. These slowly proliferating late embryonic RPs share a core transcriptional phenotype with quiescent adult forebrain NSCs. Together, these findings support a model wherein cortical RPs maintain a core transcriptional identity from embryogenesis through to adulthood and wherein the transition to a quiescent adult NSC occurs during late neurogenesis.
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http://dx.doi.org/10.1016/j.celrep.2017.12.017DOI Listing
December 2017

The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation.

Nat Commun 2017 09 11;8(1):499. Epub 2017 Sep 11.

Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada, M5G 1M1.

The capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C. albicans transcription factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit β-1,3-glucan synthesis. Cas5 has distinct transcriptional targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance.Cas5 is a transcriptional regulator of responses to cell wall stress in the fungal pathogen Candida albicans. Here, Xie et al. show that Cas5 also modulates cell cycle dynamics and responses to antifungal drugs.
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http://dx.doi.org/10.1038/s41467-017-00547-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593949PMC
September 2017

SMYD2 lysine methyltransferase regulates leukemia cell growth and regeneration after genotoxic stress.

Oncotarget 2017 Mar;8(10):16712-16727

Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2.Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.
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http://dx.doi.org/10.18632/oncotarget.15147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369996PMC
March 2017

Functional Genomic Analysis of Candida albicans Adherence Reveals a Key Role for the Arp2/3 Complex in Cell Wall Remodelling and Biofilm Formation.

PLoS Genet 2016 Nov 21;12(11):e1006452. Epub 2016 Nov 21.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Fungal biofilms are complex, structured communities that can form on surfaces such as catheters and other indwelling medical devices. Biofilms are of particular concern with Candida albicans, one of the leading opportunistic fungal pathogens of humans. C. albicans biofilms include yeast and filamentous cells that are surrounded by an extracellular matrix, and they are intrinsically resistant to antifungal drugs such that resolving biofilm infections often requires surgery to remove the contaminated device. C. albicans biofilms form through a regulated process of adhesion to surfaces, filamentation, maturation, and ultimately dispersion. To uncover new strategies to block the initial stages of biofilm formation, we utilized a functional genomic approach to identify genes that modulate C. albicans adherence. We screened a library of 1,481 double barcoded doxycycline-repressible conditional gene expression strains covering ~25% of the C. albicans genome. We identified five genes for which transcriptional repression impaired adherence, including: ARC18, PMT1, MNN9, SPT7, and orf19.831. The most severe adherence defect was observed upon transcriptional repression of ARC18, which encodes a member of the Arp2/3 complex that is involved in regulation of the actin cytoskeleton and endocytosis. Depletion of components of the Arp2/3 complex not only impaired adherence, but also caused reduced biofilm formation, increased cell surface hydrophobicity, and increased exposure of cell wall chitin and β-glucans. Reduced function of the Arp2/3 complex led to impaired cell wall integrity and activation of Rho1-mediated cell wall stress responses, thereby causing cell wall remodelling and reduced adherence. Thus, we identify important functional relationships between cell wall stress responses and a novel mechanism that controls adherence and biofilm formation, thereby illuminating novel strategies to cripple a leading fungal pathogen of humans.
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http://dx.doi.org/10.1371/journal.pgen.1006452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147769PMC
November 2016

The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation.

Cell Rep 2016 05 5;15(7):1554-1565. Epub 2016 May 5.

Department of Immunology, University of Toronto, Medical Sciences Building, Toronto, ON M5S 1A8, Canada. Electronic address:

Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR.
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http://dx.doi.org/10.1016/j.celrep.2016.04.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888775PMC
May 2016

Cytokinetic effects of Wee1 disruption in pancreatic cancer.

Cell Cycle 2016 ;15(4):593-604

a Ontario Cancer Institute/Princess Margaret Cancer Center , Toronto , Ontario , Canada.

The Wee1 kinase, which is activated in response to DNA damage, regulates exit from G2 through inhibitory phosphorylation of Cdk1/Cdc2, and is an attractive drug target. However, recent work has highlighted effects of Cdk2 phosphorylation by Wee1 on movement through S-phase, suggesting the potential to sensitize to S-phase specific agents by Wee1 inhibitors. In this paper we applied multiparametric flow cytometry to patient-derived pancreatic cancer xenograft tumor cells to study the cell cycle perturbations of Wee1 disruption via the small molecule inhibitor MK-1775, and genetic knockdown. We find that in vitro treatment with MK-1775, and to a lesser degree, Wee1 RNA transcript knockdown, results in the striking appearance of S-phase cells prematurely entering into mitosis. This effect was not seen in vivo in any of the models tested. Here, although we noted an increase of S-phase cells expressing the damage response marker γH2AX, treatment with MK-1775 did not significantly sensitize cells to the cytidine analog gemcitabine. Treatment with MK-1775 did result in a transient but large increase in cells expressing the mitotic marker phosphorylated H3S10 that reached a peak 4 hours after treatment. This suggests a role for Wee1 regulating the progression of genomically unstable cancer cells through G2 in the absence of extrinsically-applied DNA damage. A single dose of 8Gy ionizing radiation resulted in the time-dependent accumulation of Cyclin A2 positive/phosphorylated H3S10 negative cells at the 4N position, which was abrogated by treatment with MK-1775. Consistent with these findings, a genome-scale pooled RNA interference screen revealed that toxic doses of MK-1775 are suppressed by CDK2 or Cyclin A2 knockdown. These findings support G2 exit as the more significant effect of Wee1 inhibition in pancreatic cancers.
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http://dx.doi.org/10.1080/15384101.2016.1138188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5056606PMC
December 2016

Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs.

Cancer Res 2015 Oct 21;75(19):4211-23. Epub 2015 Aug 21.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada. Department of Radiation Oncology, University of Toronto, Toronto, Ontario, Canada. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. Ontario Institute for Cancer Research, Toronto, Ontario, Canada.

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-1107DOI Listing
October 2015

Inhibition of the Mitochondrial Protease ClpP as a Therapeutic Strategy for Human Acute Myeloid Leukemia.

Cancer Cell 2015 Jun;27(6):864-76

Princess Margaret Cancer Centre, Toronto, ON M5G 2M9, Canada; Department of Medicine, University of Toronto, Toronto, ON M5G 2C4, Canada.

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.
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http://dx.doi.org/10.1016/j.ccell.2015.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461837PMC
June 2015

Global analysis of fungal morphology exposes mechanisms of host cell escape.

Nat Commun 2015 Mar 31;6:6741. Epub 2015 Mar 31.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

Developmental transitions between single-cell yeast and multicellular filaments underpin virulence of diverse fungal pathogens. For the leading human fungal pathogen Candida albicans, filamentation is thought to be required for immune cell escape via induction of an inflammatory programmed cell death. Here we perform a genome-scale analysis of C. albicans morphogenesis and identify 102 negative morphogenetic regulators and 872 positive regulators, highlighting key roles for ergosterol biosynthesis and N-linked glycosylation. We demonstrate that C. albicans filamentation is not required for escape from host immune cells; instead, macrophage pyroptosis is driven by fungal cell-wall remodelling and exposure of glycosylated proteins in response to the macrophage phagosome. The capacity of killed, previously phagocytized cells to drive macrophage lysis is also observed with the distantly related fungal pathogen Cryptococcus neoformans. This study provides a global view of morphogenetic circuitry governing a key virulence trait, and illuminates a new mechanism by which fungi trigger host cell death.
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http://dx.doi.org/10.1038/ncomms7741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382923PMC
March 2015

Differential regulation of FGFR3 by PTPN1 and PTPN2.

Proteomics 2015 Jan 17;15(2-3):419-33. Epub 2014 Dec 17.

Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto, Canada.

Aberrant expression and activation of FGFR3 is associated with disease states including bone dysplasia and malignancies of bladder, cervix, and bone marrow. MS analysis of protein-phosphotyrosine in multiple myeloma cells revealed a prevalent phosphorylated motif, D/EYYR/K, derived from the kinase domain activation loops of tyrosine kinases including FGFR3 corresponding to a recognition sequence of protein-tyrosine phosphatase PTPN1. Knockdown of PTPN1 or the related enzyme PTPN2 by RNAi resulted in ligand-independent activation of FGFR3. Modulation of FGFR3 activation loop phosphorylation by both PTPN1 and PTPN2 was a function of receptor trafficking and phosphotyrosine phosphatase (PTP) compartmentalization. The FGFR3 activation loop motif DYYKK(650) is altered to DYYKE(650) in the oncogenic variant FGFR3(K650E) , and consequently it is constitutively fully activated and unaffected by activation loop phosphorylation. FGFR3(K650E) was nevertheless remarkably sensitive to negative regulation by PTPN1 and PTPN2. This suggests that in addition to modulating FGFR3 phosphorylation, PTPN1 and PTPN2 constrain the kinase domain by fostering an inactive-state. Loss of this constraint in response to ligand or impaired PTPN1/N2 may initiate FGFR3 activation. These results suggest a model wherein PTP expression levels may define conditions that select for ectopic FGFR3 expression and activation during tumorigenesis.
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http://dx.doi.org/10.1002/pmic.201400259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032629PMC
January 2015

The RhoGEF GEF-H1 is required for oncogenic RAS signaling via KSR-1.

Cancer Cell 2014 Feb;25(2):181-95

Princess Margaret Cancer Center, University Health Network, 101 College Street, Room 8-703, Toronto Medical Discovery Tower, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Medicine, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Department of Immunology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada; Division of Rheumatology, St. Michael's Hospital, 30 Bond Street, Toronto, ON M5B 1W8, Canada. Electronic address:

Cellular transformation by oncogenic RAS engages the MAPK pathway under strict regulation by the scaffold protein KSR-1. Here, we report that the guanine nucleotide exchange factor GEF-H1 plays a critical role in a positive feedback loop for the RAS/MAPK pathway independent of its RhoGEF activity. GEF-H1 acts as an adaptor protein linking the PP2A B' subunits to KSR-1, thereby mediating the dephosphorylation of KSR-1 S392 and activation of MAPK signaling. GEF-H1 is important for the growth and survival of HRAS(V12)-transformed cells and pancreatic tumor xenografts. GEF-H1 expression is induced by oncogenic RAS and is correlated with pancreatic neoplastic progression. Our results, therefore, identify GEF-H1 as an amplifier of MAPK signaling and provide mechanistic insight into the progression of RAS mutant tumors.
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http://dx.doi.org/10.1016/j.ccr.2014.01.025DOI Listing
February 2014

shRNA kinome screen identifies TBK1 as a therapeutic target for HER2+ breast cancer.

Cancer Res 2014 Apr 31;74(7):2119-30. Epub 2014 Jan 31.

Authors' Affiliations: Division of Advanced Diagnostics, Toronto General Research Institute-University Health Network; Medicinal Chemistry Platform, Ontario Institute for Cancer Research; The Donnelly Centre, University of Toronto; Program in Developmental and Stem Cell Biology, Department of Molecular Genetics, The Hospital for Sick Children; Ontario Cancer Institute, University of Toronto, and Drug Discovery Program, Department of Pharmacology and Toxicology; Toronto, Ontario, Canada.

HER2(+) breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2(+) breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that "immortalize" the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu(+) tumorsphere versus monolayer cells with a lentivirus short hairpin RNA kinome library. We identified kinases such as the mitogen-activated protein kinase and the TGFβR protein family, previously implicated in HER2(+) breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IκB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2(+) breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKε, suppressed growth of both mouse and human HER2(+) breast cancer cells. TBK1/IKKε inhibition promoted cellular senescence by suppressing p65-NF-κB and inducing p16(Ink4a). In addition, TBK1/IKKε inhibition cooperated with lapatinib, a HER2/EGFR1-targeted drug, to accelerate apoptosis and kill HER2(+) breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2(+) breast cancer may benefit from anti-TBK1/IKKε plus anti-HER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2(+) breast cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-13-2138DOI Listing
April 2014

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities.

Mol Syst Biol 2013 Oct 8;9:696. Epub 2013 Oct 8.

1] Donnelly Centre and Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada [2] Saskatchewan Cancer Agency, Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.
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http://dx.doi.org/10.1038/msb.2013.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817404PMC
October 2013

Interaction domains of Sos1/Grb2 are finely tuned for cooperative control of embryonic stem cell fate.

Cell 2013 Feb;152(5):1008-20

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada.

Metazoan evolution involves increasing protein domain complexity, but how this relates to control of biological decisions remains uncertain. The Ras guanine nucleotide exchange factor (RasGEF) Sos1 and its adaptor Grb2 are multidomain proteins that couple fibroblast growth factor (FGF) signaling to activation of the Ras-Erk pathway during mammalian development and drive embryonic stem cells toward the primitive endoderm (PrE) lineage. We show that the ability of Sos1/Grb2 to appropriately regulate pluripotency and differentiation factors and to initiate PrE development requires collective binding of multiple Sos1/Grb2 domains to their protein and phospholipid ligands. This provides a cooperative system that only allows lineage commitment when all ligand-binding domains are occupied. Furthermore, our results indicate that the interaction domains of Sos1 and Grb2 have evolved so as to bind ligands not with maximal strength but with specificities and affinities that maintain cooperativity. This optimized system ensures that PrE lineage commitment occurs in a timely and selective manner during embryogenesis.
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http://dx.doi.org/10.1016/j.cell.2013.01.056DOI Listing
February 2013

Suppression of cancer progression by MGAT1 shRNA knockdown.

PLoS One 2012 5;7(9):e43721. Epub 2012 Sep 5.

Ontario Cancer Institute, Princess Margaret Hospital, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043721PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434202PMC
February 2013

Essential gene profiles in breast, pancreatic, and ovarian cancer cells.

Cancer Discov 2012 Feb 29;2(2):172-189. Epub 2011 Dec 29.

Donnelly Centre and Banting & Best Department of Medical Research, University of Toronto, Toronto, Canada.

Unlabelled: 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5057396PMC
http://dx.doi.org/10.1158/2159-8290.CD-11-0224DOI Listing
February 2012

A genome wide shRNA screen identifies α/β hydrolase domain containing 4 (ABHD4) as a novel regulator of anoikis resistance.

Apoptosis 2012 Jul;17(7):666-78

Ontario Cancer Institute, Princess Margaret Hospital, Toronto, Canada.

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.
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http://dx.doi.org/10.1007/s10495-012-0723-4DOI Listing
July 2012

Analysis of early C2C12 myogenesis identifies stably and differentially expressed transcriptional regulators whose knock-down inhibits myoblast differentiation.

Physiol Genomics 2012 Feb 6;44(2):183-97. Epub 2011 Dec 6.

Department of Human Genetics, McGill University, Canada.

Myogenesis is a tightly controlled process involving the transcriptional activation and repression of thousands of genes. Although many components of the transcriptional network regulating the later phases of myogenesis have been identified, relatively few studies have described the transcriptional landscape during the first 24 h, when myoblasts commit to differentiate. Through dense temporal profiling of differentiating C2C12 myoblasts, we identify 193 transcriptional regulators (TRs) whose expression is significantly altered within the first 24 h of myogenesis. A high-content shRNA screen of 77 TRs involving 427 stable lines identified 42 genes whose knockdown significantly inhibits differentiation of C2C12 myoblasts. Of the TRs that were differentially expressed within the first 24 h, over half inhibited differentiation when knocked down, including known regulators of myogenesis (Myod1, Myog, and Myf5), as well as 19 TRs not previously associated with this process. Surprisingly, a similar proportion (55%) of shRNAs targeting TRs whose expression did not change also inhibited C2C12 myogenesis. We further show that a subset of these TRs inhibits myogenesis by downregulating expression of known regulatory and structural proteins. Our findings clearly illustrate that several TRs critical for C2C12 myogenesis are not differentially regulated, suggesting that approaches that focus functional studies on differentially-expressed transcripts will fail to provide a comprehensive view of this complex process.
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http://dx.doi.org/10.1152/physiolgenomics.00093.2011DOI Listing
February 2012

COLT-Cancer: functional genetic screening resource for essential genes in human cancer cell lines.

Nucleic Acids Res 2012 Jan 18;40(Database issue):D957-63. Epub 2011 Nov 18.

Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 160 College St, Toronto, ON M5S 3E1, Canada M5G 0A3.

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.
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http://dx.doi.org/10.1093/nar/gkr959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3245009PMC
January 2012
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