Publications by authors named "Stephane Angers"

92 Publications

Wnt signaling inhibition confers induced synthetic lethality to PARP inhibitors.

Authors:
Stephane Angers

EMBO Mol Med 2021 Apr 30;13(4):e14002. Epub 2021 Mar 30.

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada.

Despite considerable efforts, therapeutic strategies targeting the Wnt pathway are still not clinically available. The pervasive role of Wnt-βcatenin signaling for the control of stem cells during normal tissue homeostasis makes the on-target toxicity of available therapeutic grade molecules an important limitation preventing their clinical introduction. The article in this issue of EMBO Molecular Medicine by Kaur et al (2021) reveals that treatment of Wnt-addicted cancer cells with inhibitors of Wnt signaling induces a state of BRCAness leading to hypersensitivity to PARP inhibitors. This is a new example of induced synthetic lethality that could pave the way for new indications for PARP inhibitors or may contribute to the long-awaited clinical introduction of therapeutic agents targeting the Wnt pathway.
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http://dx.doi.org/10.15252/emmm.202114002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033510PMC
April 2021

The RanBP2/RanGAP1-SUMO complex gates β-arrestin2 nuclear entry to regulate the Mdm2-p53 signaling axis.

Oncogene 2021 Mar 1;40(12):2243-2257. Epub 2021 Mar 1.

Inserm, U1016, Institut Cochin, Paris, France.

Mdm2 antagonizes the tumor suppressor p53. Targeting the Mdm2-p53 interaction represents an attractive approach for the treatment of cancers with functional p53. Investigating mechanisms underlying Mdm2-p53 regulation is therefore important. The scaffold protein β-arrestin2 (β-arr2) regulates tumor suppressor p53 by counteracting Mdm2. β-arr2 nucleocytoplasmic shuttling displaces Mdm2 from the nucleus to the cytoplasm resulting in enhanced p53 signaling. β-arr2 is constitutively exported from the nucleus, via a nuclear export signal, but mechanisms regulating its nuclear entry are not completely elucidated. β-arr2 can be SUMOylated, but no information is available on how SUMO may regulate β-arr2 nucleocytoplasmic shuttling. While we found β-arr2 SUMOylation to be dispensable for nuclear import, we identified a non-covalent interaction between SUMO and β-arr2, via a SUMO interaction motif (SIM), that is required for β-arr2 cytonuclear trafficking. This SIM promotes association of β-arr2 with the multimolecular RanBP2/RanGAP1-SUMO nucleocytoplasmic transport hub that resides on the cytoplasmic filaments of the nuclear pore complex. Depletion of RanBP2/RanGAP1-SUMO levels result in defective β-arr2 nuclear entry. Mutation of the SIM inhibits β-arr2 nuclear import, its ability to delocalize Mdm2 from the nucleus to the cytoplasm and enhanced p53 signaling in lung and breast tumor cell lines. Thus, a β-arr2 SIM nuclear entry checkpoint, coupled with active β-arr2 nuclear export, regulates its cytonuclear trafficking function to control the Mdm2-p53 signaling axis.
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http://dx.doi.org/10.1038/s41388-021-01704-wDOI Listing
March 2021

Single-cell chromatin accessibility profiling of glioblastoma identifies an invasive cancer stem cell population associated with lower survival.

Elife 2021 Jan 11;10. Epub 2021 Jan 11.

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

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.
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http://dx.doi.org/10.7554/eLife.64090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847307PMC
January 2021

Copper bioavailability is a KRAS-specific vulnerability in colorectal cancer.

Nat Commun 2020 07 24;11(1):3701. Epub 2020 Jul 24.

Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montreal, 2950, Chemin de la Polytechnique, Montréal, QC, H3T 1J4, Canada.

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.
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http://dx.doi.org/10.1038/s41467-020-17549-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7381612PMC
July 2020

Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation.

Proc Natl Acad Sci U S A 2020 07 29;117(28):16690-16701. Epub 2020 Jun 29.

Department of Systems Biology, Harvard Medical School, Boston, MA 02115;

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/μm to 0.54/μm Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.
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http://dx.doi.org/10.1073/pnas.1910547117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368285PMC
July 2020

Precise Temporal Regulation of Post-transcriptional Repressors Is Required for an Orderly Drosophila Maternal-to-Zygotic Transition.

Cell Rep 2020 06;31(12):107783

Department of Molecular Genetics, University of Toronto, 661 University Avenue, Toronto, ON M5G 1M1, Canada. Electronic address:

In animal embryos, the maternal-to-zygotic transition (MZT) hands developmental control from maternal to zygotic gene products. We show that the maternal proteome represents more than half of the protein-coding capacity of Drosophila melanogaster's genome, and that 2% of this proteome is rapidly degraded during the MZT. Cleared proteins include the post-transcriptional repressors Cup, Trailer hitch (TRAL), Maternal expression at 31B (ME31B), and Smaug (SMG). Although the ubiquitin-proteasome system is necessary for clearance of these repressors, distinct E3 ligase complexes target them: the C-terminal to Lis1 Homology (CTLH) complex targets Cup, TRAL, and ME31B for degradation early in the MZT and the Skp/Cullin/F-box-containing (SCF) complex targets SMG at the end of the MZT. Deleting the C-terminal 233 amino acids of SMG abrogates F-box protein interaction and confers immunity to degradation. Persistent SMG downregulates zygotic re-expression of mRNAs whose maternal contribution is degraded by SMG. Thus, clearance of SMG permits an orderly MZT.
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http://dx.doi.org/10.1016/j.celrep.2020.107783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372737PMC
June 2020

Metabolic Regulation of the Epigenome Drives Lethal Infantile Ependymoma.

Cell 2020 06 22;181(6):1329-1345.e24. Epub 2020 May 22.

The Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Developmental & Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5G 1L7, Canada.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
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http://dx.doi.org/10.1016/j.cell.2020.04.047DOI Listing
June 2020

Nanostructured Architectures Promote the Mesenchymal-Epithelial Transition for Invasive Cells.

ACS Nano 2020 05 11;14(5):5324-5336. Epub 2020 May 11.

Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, M5S 3G9, Canada.

Dynamic modulation of cellular phenotypes between the epithelial and mesenchymal states-the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET)-plays an important role in cancer progression. Nanoscale topography of culture substrates is known to affect the migration and EMT of cancer cells. However, existing platforms heavily rely on simple geometries such as grooved lines or cylindrical post arrays, which may oversimplify the complex interaction between cells and nanotopography . Here, we use electrodeposition to construct finely controlled surfaces with biomimetic fractal nanostructures as a means of examining the roles of nanotopography during the EMT/MET process. We found that nanostructures in the size range of 100 to 500 nm significantly promote MET for invasive breast and prostate cancer cells. The "METed" cells acquired distinct expression of epithelial and mesenchymal markers, displayed perturbed morphologies, and exhibited diminished migration and invasion, even after the removal of a nanotopographical stimulus. The phosphorylation of GSK-3 was decreased, which further tuned the expression of Snail and modulated the EMT/MET process. Our findings suggest that invasive cancer cells respond to the geometries and dimensions of complex nanostructured architectures.
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http://dx.doi.org/10.1021/acsnano.9b07350DOI Listing
May 2020

The RNA-Binding Protein Rasputin/G3BP Enhances the Stability and Translation of Its Target mRNAs.

Cell Rep 2020 03;30(10):3353-3367.e7

Department of Molecular Genetics, University of Toronto, 661 University Avenue, Toronto, ON M5G 1M1, Canada. Electronic address:

G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules.
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http://dx.doi.org/10.1016/j.celrep.2020.02.066DOI Listing
March 2020

IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers.

J Cell Biol 2020 02;219(2)

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.
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http://dx.doi.org/10.1083/jcb.201903017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041691PMC
February 2020

Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.

Cell 2019 11 21;179(6):1330-1341.e13. Epub 2019 Nov 21.

Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Case Comprehensive Cancer Center, Cleveland, OH 44106, USA. Electronic address:

Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.
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http://dx.doi.org/10.1016/j.cell.2019.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241652PMC
November 2019

High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.

Nat Biomed Eng 2019 10 23;3(10):796-805. Epub 2019 Sep 23.

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 10 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.
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http://dx.doi.org/10.1038/s41551-019-0454-8DOI Listing
October 2019

Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

Elife 2019 08 27;8. Epub 2019 Aug 27.

Donnelly Centre, University of Toronto, Toronto, Canada.

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.
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http://dx.doi.org/10.7554/eLife.46134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711705PMC
August 2019

Identifying chemogenetic interactions from CRISPR screens with drugZ.

Genome Med 2019 08 22;11(1):52. Epub 2019 Aug 22.

Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: Chemogenetic profiling enables the identification of gene mutations that enhance or suppress the activity of chemical compounds. This knowledge provides insights into drug mechanism of action, genetic vulnerabilities, and resistance mechanisms, all of which may help stratify patient populations and improve drug efficacy. CRISPR-based screening enables sensitive detection of drug-gene interactions directly in human cells, but until recently has primarily been used to screen only for resistance mechanisms.

Results: We present drugZ, an algorithm for identifying both synergistic and suppressor chemogenetic interactions from CRISPR screens. DrugZ identifies synthetic lethal interactions between PARP inhibitors and both known and novel members of the DNA damage repair pathway, confirms KEAP1 loss as a resistance factor for ERK inhibitors in oncogenic KRAS backgrounds, and defines the genetic context for temozolomide activity.

Conclusions: DrugZ is an open-source Python software for the analysis of genome-scale drug modifier screens. The software accurately identifies genetic perturbations that enhance or suppress drug activity. Interestingly, analysis of new and previously published data reveals tumor suppressor genes are drug-agnostic resistance genes in drug modifier screens. The software is available at github.com/hart-lab/drugz .
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http://dx.doi.org/10.1186/s13073-019-0665-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706933PMC
August 2019

Genome-Wide CRISPR-Cas9 Screens Expose Genetic Vulnerabilities and Mechanisms of Temozolomide Sensitivity in Glioblastoma Stem Cells.

Cell Rep 2019 04;27(3):971-986.e9

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON, Canada; Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto, ON, Canada. Electronic address:

Glioblastoma therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells (GSCs) to interrogate function of the coding genome, we identify actionable pathways responsible for growth, which reveal the gene-essential circuitry of GBM stemness and proliferation. In particular, we characterize members of the SOX transcription factor family, SOCS3, USP8, and DOT1L, and protein ufmylation as important for GSC growth. Additionally, we reveal mechanisms of temozolomide resistance that could lead to combination strategies. By reaching beyond static genome analysis of bulk tumors, with a genome-wide functional approach, we reveal genetic dependencies within a broad range of biological processes to provide increased understanding of GBM growth and treatment resistance.
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http://dx.doi.org/10.1016/j.celrep.2019.03.047DOI Listing
April 2019

Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies.

Proc Natl Acad Sci U S A 2019 04 20;116(14):6812-6817. Epub 2019 Mar 20.

Program in Molecular Medicine, Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada;

Aberrant activation of Wnt/β-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.
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http://dx.doi.org/10.1073/pnas.1817246116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452705PMC
April 2019

Wnt and Notch signaling govern self-renewal and differentiation in a subset of human glioblastoma stem cells.

Genes Dev 2019 05 6;33(9-10):498-510. Epub 2019 Mar 6.

Developmental and Stem Cell Biology Program, Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.

Developmental signal transduction pathways act diversely, with context-dependent roles across systems and disease types. Glioblastomas (GBMs), which are the poorest prognosis primary brain cancers, strongly resemble developmental systems, but these growth processes have not been exploited therapeutically, likely in part due to the extreme cellular and genetic heterogeneity observed in these tumors. The role of Wnt/βcatenin signaling in GBM stem cell (GSC) renewal and fate decisions remains controversial. Here, we report context-specific actions of Wnt/βcatenin signaling in directing cellular fate specification and renewal. A subset of primary GBM-derived stem cells requires Wnt proteins for self-renewal, and this subset specifically relies on Wnt/βcatenin signaling for enhanced tumor burden in xenograft models. In an orthotopic Wnt reporter model, Wnt GBM cells (which exhibit high levels of βcatenin signaling) are a faster-cycling, highly self-renewing stem cell pool. In contrast, Wnt cells (with low levels of signaling) are slower cycling and have decreased self-renewing potential. Dual inhibition of Wnt/βcatenin and Notch signaling in GSCs that express high levels of the proneural transcription factor leads to robust neuronal differentiation and inhibits clonogenic potential. Our work identifies new contexts for Wnt modulation for targeting stem cell differentiation and self-renewal in GBM heterogeneity, which deserve further exploration therapeutically.
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http://dx.doi.org/10.1101/gad.321968.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499328PMC
May 2019

Agonist-induced desensitisation of β -adrenoceptors: Where, when, and how?

Br J Pharmacol 2019 07 7;176(14):2539-2558. Epub 2019 Apr 7.

Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.

β -Adrenoceptor agonists have proven useful in the treatment of overactive bladder syndrome, but it is not known whether their efficacy during chronic administration may be limited by receptor-induced desensitisation. Whereas the β -adrenoceptor has phosphorylation sites that are important for desensitisation, the β -adrenoceptor lacks these; therefore, it had been assumed that β -adrenoceptors are largely resistant to agonist-induced desensitisation. While all direct comparative studies demonstrate that β -adrenoceptors are less susceptible to desensitisation than β -adrenoceptors, desensitisation of β -adrenoceptors has been observed in many models and treatment settings. Chimeric β - and β -adrenoceptors have demonstrated that the C-terminal tail of the receptor plays an important role in the relative resistance to desensitisation but is not the only relevant factor. While the evidence from some models, such as transfected CHO cells, is inconsistent, it appears that desensitisation is observed more often after long-term (hours to days) than short-term (minutes to hours) agonist exposure. When it occurs, desensitisation of β -adrenoceptors can involve multiple levels including down-regulation of its mRNA and the receptor protein and alterations in post-receptor signalling events. The relative contributions of these mechanistic factors apparently depend on the cell type under investigation. Which if any of these factors is applicable to the human urinary bladder remains to be determined. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
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http://dx.doi.org/10.1111/bph.14633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592865PMC
July 2019

ARGLU1 is a transcriptional coactivator and splicing regulator important for stress hormone signaling and development.

Nucleic Acids Res 2019 04;47(6):2856-2870

Department of Pharmaceutical Sciences, University of Toronto, Toronto, ON M5S 3M2, Canada.

Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.
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http://dx.doi.org/10.1093/nar/gkz010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451108PMC
April 2019

Dual Regulatory Functions of SUFU and Targetome of GLI2 in SHH Subgroup Medulloblastoma.

Dev Cell 2019 01 13;48(2):167-183.e5. Epub 2018 Dec 13.

Program in Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Joint Institute of Genetics and Genomic Medicine, Zhejiang University and University of Toronto, Toronto, ON, Canada. Electronic address:

SUFU alterations are common in human Sonic Hedgehog (SHH) subgroup medulloblastoma (MB). However, its tumorigenic mechanisms have remained elusive. Here, we report that loss of Sufu alone is unable to induce MB formation in mice, due to insufficient Gli2 activation. Simultaneous loss of Spop, an E3 ubiquitin ligase targeting Gli2, restores robust Gli2 activation and induces rapid MB formation in Sufu knockout background. We also demonstrated a tumor-promoting role of Sufu in Smo-activated MB (∼60% of human SHH MB) by maintaining robust Gli activity. Having established Gli2 activation as a key driver of SHH MB, we report a comprehensive analysis of its targetome. Furthermore, we identified Atoh1 as a target and molecular accomplice of Gli2 that activates core SHH MB signature genes in a synergistic manner. Overall, our work establishes the dual role of SUFU in SHH MB and provides mechanistic insights into transcriptional regulation underlying Gli2-mediated SHH MB tumorigenesis.
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http://dx.doi.org/10.1016/j.devcel.2018.11.015DOI Listing
January 2019

Three-Dimensional Nanostructured Architectures Enable Efficient Neural Differentiation of Mesenchymal Stem Cells via Mechanotransduction.

Nano Lett 2018 11 18;18(11):7188-7193. Epub 2018 Oct 18.

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy , University of Toronto , Toronto , M5S 3M2 , Canada.

Cell morphology and geometry affect cellular processes such as stem cell differentiation, suggesting that these parameters serve as fundamental regulators of biological processes within the cell. Hierarchical architectures featuring micro- and nanotopographical features therefore offer programmable systems for stem cell differentiation. However, a limited number of studies have explored the effects of hierarchical architectures due to the complexity of fabricating systems with rationally tunable micro- and nanostructuring. Here, we report three-dimensional (3D) nanostructured microarchitectures that efficiently regulate the fate of human mesenchymal stem cells (hMSCs). These nanostructured architectures strongly promote cell alignment and efficient neurogenic differentiation where over 85% of hMSCs express microtubule-associated protein 2 (MAP2), a mature neural marker, after 7 days of culture on the nanostructured surface. Remarkably, we found that the surface morphology of nanostructured surface is a key factor that promotes neurogenesis and that highly spiky structures promote more efficient neuronal differentiation. Immunostaining and gene expression profiling revealed significant upregulation of neuronal markers compared to unpatterned surfaces. These findings suggest that the 3D nanostructured microarchitectures can play a critical role in defining stem cell behavior.
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http://dx.doi.org/10.1021/acs.nanolett.8b03313DOI Listing
November 2018

A synthetic anti-Frizzled antibody engineered for broadened specificity exhibits enhanced anti-tumor properties.

MAbs 2018 Nov-Dec;10(8):1157-1167. Epub 2018 Sep 25.

a Terrence Donnelly Centre for Cellular and Biomolecular Research , University of Toronto , Toronto , Canada.

Secreted Wnt ligands play a major role in the development and progression of many cancers by modulating signaling through cell-surface Frizzled receptors (FZDs). In order to achieve maximal effect on Wnt signaling by targeting the cell surface, we developed a synthetic antibody targeting six of the 10 human FZDs. We first identified an anti-FZD antagonist antibody (F2) with a specificity profile matching that of OMP-18R5, a monoclonal antibody that inhibits growth of many cancers by targeting FZD7, FZD1, FZD2, FZD5 and FZD8. We then used combinatorial antibody engineering by phage display to develop a variant antibody F2.A with specificity broadened to include FZD4. We confirmed that F2.A blocked binding of Wnt ligands, but not binding of Norrin, a ligand that also activates FZD4. Importantly, F2.A proved to be much more efficacious than either OMP-18R5 or F2 in inhibiting the growth of multiple RNF43-mutant pancreatic ductal adenocarcinoma cell lines, including patient-derived cells.
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http://dx.doi.org/10.1080/19420862.2018.1515565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284576PMC
June 2019

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

Nature 2018 07 4;559(7713):285-289. Epub 2018 Jul 4.

The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.
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http://dx.doi.org/10.1038/s41586-018-0291-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071917PMC
July 2018

Wnt signaling in development and tissue homeostasis.

Development 2018 06 8;145(11). Epub 2018 Jun 8.

University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada

The Wnt-β-catenin signaling pathway is an evolutionarily conserved cell-cell communication system that is important for stem cell renewal, cell proliferation and cell differentiation both during embryogenesis and during adult tissue homeostasis. Genetic or epigenetic events leading to hypo- or hyper-activation of the Wnt-β-catenin signaling cascade have also been associated with human diseases such as cancer. Understanding how this pathway functions is thus integral for developing therapies to treat diseases or for regenerative medicine approaches. Here, and in the accompanying poster, we provide an overview of Wnt-β-catenin signaling and briefly highlight its key functions during development and adult tissue homeostasis.
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http://dx.doi.org/10.1242/dev.146589DOI Listing
June 2018

Publisher Correction: A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Nat Chem Biol 2018 Sep;14(9):902

Department of Early Discovery Biochemistry, Genentech, South San Francisco, CA, USA.

The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41589-018-0069-5DOI Listing
September 2018

A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Nat Chem Biol 2018 06 9;14(6):582-590. Epub 2018 Apr 9.

Department of Early Discovery Biochemistry, Genentech, South San Francisco, CA, USA.

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5 intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.
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http://dx.doi.org/10.1038/s41589-018-0035-2DOI Listing
June 2018

High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies.

Mol Cell 2018 02 25;69(3):517-532.e11. Epub 2018 Jan 25.

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. Electronic address:

mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.
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http://dx.doi.org/10.1016/j.molcel.2017.12.020DOI Listing
February 2018