Publications by authors named "Graham MacLeod"

14 Publications

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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

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

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

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

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

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

SAPCD2 Controls Spindle Orientation and Asymmetric Divisions by Negatively Regulating the Gαi-LGN-NuMA Ternary Complex.

Dev Cell 2016 Jan;36(1):50-62

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, ON M5S 3M2, Canada; Department of Biochemistry, University of Toronto, ON M5S 1A8, Canada. Electronic address:

Control of cell-division orientation is integral to epithelial morphogenesis and asymmetric cell division. Proper spatiotemporal localization of the evolutionarily conserved Gαi-LGN-NuMA protein complex is critical for mitotic spindle orientation, but how this is achieved remains unclear. Here we identify Suppressor APC domain containing 2 (SAPCD2) as a previously unreported LGN-interacting protein. We show that SAPCD2 is essential to instruct planar mitotic spindle orientation in both epithelial cell cultures and mouse retinal progenitor cells in vivo. Loss of SAPCD2 randomizes spindle orientation, which in turn disrupts cyst morphogenesis in three-dimensional cultures, and triples the number of terminal asymmetric cell divisions in the developing retina. Mechanistically, we show that SAPCD2 negatively regulates the localization of LGN at the cell cortex, likely by competing with NuMA for its binding. These results uncover SAPCD2 as a key regulator of the ternary complex controlling spindle orientation during morphogenesis and asymmetric cell divisions.
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http://dx.doi.org/10.1016/j.devcel.2015.12.016DOI Listing
January 2016

High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.

Cell 2015 Dec 25;163(6):1515-26. Epub 2015 Nov 25.

Donnelly Centre, 160 College Street, Toronto, ON M5S3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S1A1, Canada; Canadian Institute for Advanced Research, Toronto, ON M5G1Z8, Canada. Electronic address:

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.
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http://dx.doi.org/10.1016/j.cell.2015.11.015DOI Listing
December 2015

Positive regulation of TRAF6-dependent innate immune responses by protein phosphatase PP1-γ.

PLoS One 2014 20;9(2):e89284. Epub 2014 Feb 20.

Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

Innate immune sensors such as Toll-like receptors (TLRs) differentially utilize adaptor proteins and additional molecular mediators to ensure robust and precise immune responses to pathogen challenge. Through a gain-of-function genetic screen, we identified the gamma catalytic subunit of protein phosphatase 1 (PP1-γ) as a positive regulator of MyD88-dependent proinflammatory innate immune activation. PP1-γ physically interacts with the E3 ubiquitin ligase TRAF6, and enhances the activity of TRAF6 towards itself and substrates such as IKKγ, whereas enzymatically inactive PP1-γ represses these events. Importantly, these activities were found to be critical for cellular innate responses to pathogen challenge and microbial clearance in both mouse macrophages and human monocyte lines. These data indicate that PP1-γ phosphatase activity regulates overall TRAF6 E3 ubiquitin ligase function and promotes NF-κB-mediated innate signaling responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089284PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930702PMC
January 2015

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis.

Reproduction 2014 Jan 16;147(1):1-12. Epub 2013 Nov 16.

Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada M5S 3G5.

The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.
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http://dx.doi.org/10.1530/REP-13-0224DOI Listing
January 2014

The application of proteomic approaches to the study of mammalian spermatogenesis and sperm function.

FEBS J 2013 Nov 2;280(22):5635-51. Epub 2013 Sep 2.

Department of Cell & Systems Biology, University of Toronto, ON, Canada.

Spermatogenesis is the process by which terminally differentiated sperm are produced from male germline stem cells. This complex developmental process requires the coordination of both somatic and germ cells through phases of proliferation, meiosis, and morphological differentiation, to produce the cell responsible for the delivery of the paternal genome. With infertility affecting ~ 15% of all couples, furthering our understanding of spermatogenesis and sperm function is vital for improving the diagnosis and treatment of male factor infertility. The emerging use of proteomic technologies has played an instrumental role in our understanding of spermatogenesis by providing information regarding the genes involved. This article reviews existing proteomic literature regarding spermatogenesis and sperm function, including the proteomic characterization of spermatogenic cell types, subcellular proteomics, post-translational modifications, interactomes, and clinical studies. Future directions in the application of proteomics to the study of spermatogenesis and sperm function are also discussed.
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http://dx.doi.org/10.1111/febs.12461DOI Listing
November 2013

Tandem affinity purification in transgenic mouse embryonic stem cells identifies DDOST as a novel PPP1CC2 interacting protein.

Biochemistry 2012 Dec 16;51(48):9678-88. Epub 2012 Nov 16.

Department of Cell & Systems Biology, University of Toronto, Toronto, Ontario, Canada.

Members of the PP1 family of protein phosphatases achieve functional diversity through numerous and varied protein-protein interactions. In mammals, there are four PP1 isoforms, the ubiquitously expressed PPP1CA, PPP1CB, and PPP1CC1, and the testis specific splice isoform PPP1CC2. When the mouse Ppp1cc gene is deleted, the only phenotypic consequence is a failure of spermatogenesis in homozygous males. To elucidate the function of the Ppp1cc gene, we sought to identify novel protein-protein interactions. To this end, we have created SBP-3XFLAG-PPP1CC1 and SBP-3XFLAG-PPP1CC2 knock-in mouse embryonic stem cell lines using a gene-trap-based system. Tandem affinity purification using our knock-in cell lines identified 11 significant protein-protein interactions, including nine known PP1 interacting proteins and two additional proteins (ATP5C1 and DDOST). Reciprocal in vitro sedimentation assays confirmed the interaction between PPP1CC2 and DDOST that may have physiological implications in spermatogenesis. Immunolocalization studies revealed that DDOST localized to the nuclear envelope in dissociated spermatogenic cells and persists throughout spermatogenesis. The knock-in system described in this paper can be applied in creating tandem affinity-tagged knock-in embryonic stem cell lines with any gene for which a compatible gene-trap line is available.
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http://dx.doi.org/10.1021/bi3010158DOI Listing
December 2012

Identification of potentially damaging amino acid substitutions leading to human male infertility.

Biol Reprod 2009 Aug 15;81(2):319-26. Epub 2009 Apr 15.

Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada.

There are a number of known genetic alterations found in men with nonobstructive azoospermia, or testicular failure, such as Y microdeletions and cytogenetic abnormalities. However, the etiology of nonobstructive azoospermia is unknown in the majority of men. The aim of this study was to investigate the possibility that unexplained cases of nonobstructive azoospermia are caused by nonsynonymous single-nucleotide polymorphisms (SNPs) in the coding regions of autosomal genes associated with sperm production and fertility. Using a candidate gene approach based on genetics of male infertility in mice, we resequenced nine autosomal genes from 78 infertile men displaying testicular failure using custom-made next-generation resequencing chips. Analysis of the data revealed several novel heterozygous nonsynonymous SNPs in four of nine sequenced genes in 14 of 78 infertile men. Eight SNPs in SBF1, three SNPs in LIMK2, two SNPs in LIPE, and one SNP in TBPL1 were identified. All of the novel mutations were in a heterozygous configuration, suggesting that they may be de novo mutations with dominant negative properties.
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http://dx.doi.org/10.1095/biolreprod.109.076000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2849822PMC
August 2009