Publications by authors named "Gary D Bader"

213 Publications

A roadmap for the Human Developmental Cell Atlas.

Nature 2021 09 8;597(7875):196-205. Epub 2021 Sep 8.

Wellcome Sanger Institute, Hinxton, UK.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.
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http://dx.doi.org/10.1038/s41586-021-03620-1DOI Listing
September 2021

A clinically applicable integrative molecular classification of meningiomas.

Nature 2021 09 25;597(7874):119-125. Epub 2021 Aug 25.

MacFeeters Hamilton Neuro-Oncology Program, Princess Margaret Cancer Centre, University Health Network and University of Toronto, Toronto, Ontario, Canada.

Meningiomas are the most common primary intracranial tumour in adults. Patients with symptoms are generally treated with surgery as there are no effective medical therapies. The World Health Organization histopathological grade of the tumour and the extent of resection at surgery (Simpson grade) are associated with the recurrence of disease; however, they do not accurately reflect the clinical behaviour of all meningiomas. Molecular classifications of meningioma that reliably reflect tumour behaviour and inform on therapies are required. Here we introduce four consensus molecular groups of meningioma by combining DNA somatic copy-number aberrations, DNA somatic point mutations, DNA methylation and messenger RNA abundance in a unified analysis. These molecular groups more accurately predicted clinical outcomes compared with existing classification schemes. Each molecular group showed distinctive and prototypical biology (immunogenic, benign NF2 wild-type, hypermetabolic and proliferative) that informed therapeutic options. Proteogenomic characterization reinforced the robustness of the newly defined molecular groups and uncovered highly abundant and group-specific protein targets that we validated using immunohistochemistry. Single-cell RNA sequencing revealed inter-individual variations in meningioma as well as variations in intrinsic expression programs in neoplastic cells that mirrored the biology of the molecular groups identified.
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http://dx.doi.org/10.1038/s41586-021-03850-3DOI Listing
September 2021

Nicotinamide phosphoribosyltransferase inhibitors selectively induce apoptosis of AML stem cells by disrupting lipid homeostasis.

Cell Stem Cell 2021 Jul 12. Epub 2021 Jul 12.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Department of Medicine, University of Toronto, ON, Canada; Division of Medical Oncology and Hematology, Department of Medicine, University Health Network, Toronto, ON, Canada. Electronic address:

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.
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http://dx.doi.org/10.1016/j.stem.2021.06.004DOI Listing
July 2021

Control of tissue development and cell diversity by cell cycle-dependent transcriptional filtering.

Elife 2021 07 2;10. Epub 2021 Jul 2.

The Donnelly Centre, University of Toronto, Toronto, Canada.

Cell cycle duration changes dramatically during development, starting out fast to generate cells quickly and slowing down over time as the organism matures. The cell cycle can also act as a transcriptional filter to control the expression of long gene transcripts, which are partially transcribed in short cycles. Using mathematical simulations of cell proliferation, we identify an emergent property that this filter can act as a tuning knob to control gene transcript expression, cell diversity, and the number and proportion of different cell types in a tissue. Our predictions are supported by comparison to single-cell RNA-seq data captured over embryonic development. Additionally, evolutionary genome analysis shows that fast-developing organisms have a narrow genomic distribution of gene lengths while slower developers have an expanded number of long genes. Our results support the idea that cell cycle dynamics may be important across multicellular animals for controlling gene transcript expression and cell fate.
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http://dx.doi.org/10.7554/eLife.64951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279763PMC
July 2021

Surveilling and Tracking COVID-19 Patients Using a Portable Quantum Dot Smartphone Device.

Nano Lett 2021 06 10;21(12):5209-5216. Epub 2021 Jun 10.

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada.

The ability to rapidly diagnose, track, and disseminate information for SARS-CoV-2 is critical to minimize its spread. Here, we engineered a portable smartphone-based quantum barcode serological assay device for real-time surveillance of patients infected with SARS-CoV-2. Our device achieved a clinical sensitivity of 90% and specificity of 100% for SARS-CoV-2, as compared to 34% and 100%, respectively, for lateral flow assays in a head-to-head comparison. The lateral flow assay misdiagnosed ∼2 out of 3 SARS-CoV-2 positive patients. Our quantum dot barcode device has ∼3 times greater clinical sensitivity because it is ∼140 times more analytically sensitive than lateral flow assays. Our device can diagnose SARS-CoV-2 at different sampling dates and infectious severity. We developed a databasing app to provide instantaneous results to inform patients, physicians, and public health agencies. This assay and device enable real-time surveillance of SARS-CoV-2 seroprevalence and potential immunity.
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http://dx.doi.org/10.1021/acs.nanolett.1c01280DOI Listing
June 2021

Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells.

Nat Commun 2021 05 26;12(1):3155. Epub 2021 May 26.

McEwen Stem Cell Institute, University Health Network, Toronto, ON, Canada.

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.
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http://dx.doi.org/10.1038/s41467-021-23329-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155185PMC
May 2021

Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods.

Nat Protoc 2021 06 24;16(6):2749-2764. Epub 2021 May 24.

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

Single-cell transcriptomics can profile thousands of cells in a single experiment and identify novel cell types, states and dynamics in a wide variety of tissues and organisms. Standard experimental protocols and analysis workflows have been developed to create single-cell transcriptomic maps from tissues. This tutorial focuses on how to interpret these data to identify cell types, states and other biologically relevant patterns with the objective of creating an annotated map of cells. We recommend a three-step workflow including automatic cell annotation (wherever possible), manual cell annotation and verification. Frequently encountered challenges are discussed, as well as strategies to address them. Guiding principles and specific recommendations for software tools and resources that can be used for each step are covered, and an R notebook is included to help run the recommended workflow. Basic familiarity with computer software is assumed, and basic knowledge of programming (e.g., in the R language) is recommended.
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http://dx.doi.org/10.1038/s41596-021-00534-0DOI Listing
June 2021

SBGN Bricks Ontology as a tool to describe recurring concepts in molecular networks.

Brief Bioinform 2021 Sep;22(5)

European Institute for Systems Biology and Medicine, CIRI UMR5308, CNRS-ENS-UCBL-INSERM, Université de Lyon, 50 Avenue Tony Garnier, 69007 Lyon, France.

A comprehensible representation of a molecular network is key to communicating and understanding scientific results in systems biology. The Systems Biology Graphical Notation (SBGN) has emerged as the main standard to represent such networks graphically. It has been implemented by different software tools, and is now largely used to communicate maps in scientific publications. However, learning the standard, and using it to build large maps, can be tedious. Moreover, SBGN maps are not grounded on a formal semantic layer and therefore do not enable formal analysis. Here, we introduce a new set of patterns representing recurring concepts encountered in molecular networks, called SBGN bricks. The bricks are structured in a new ontology, the Bricks Ontology (BKO), to define clear semantics for each of the biological concepts they represent. We show the usefulness of the bricks and BKO for both the template-based construction and the semantic annotation of molecular networks. The SBGN bricks and BKO can be freely explored and downloaded at sbgnbricks.org.
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http://dx.doi.org/10.1093/bib/bbab049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425392PMC
September 2021

Distinct DNA methylation patterns associated with treatment resistance in metastatic castration resistant prostate cancer.

Sci Rep 2021 Mar 23;11(1):6630. Epub 2021 Mar 23.

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, 60 Murray Street, Toronto, ON, M5T 3L9, Canada.

Androgens are a major driver of prostate cancer (PCa) and continue to be a critical treatment target for advanced disease, which includes castration therapy and antiandrogens. However, resistance to these therapies leading to metastatic castration-resistant prostate cancer (mCRPC), and the emergence of treatment-induced neuroendocrine disease (tNEPC) remains an ongoing challenge. Instability of the DNA methylome is well established as a major hallmark of PCa development and progression. Therefore, investigating the dynamics of the methylation changes going from the castration sensitive to the tNEPC state would provide insights into novel mechanisms of resistance. Using an established xenograft model of CRPC, genome-wide methylation analysis was performed on cell lines representing various stages of PCa progression. We confirmed extensive methylation changes with the development of CRPC and tNEPC using this model. This included key genes and pathways associated with cellular differentiation and neurodevelopment. Combined analysis of methylation and gene expression changes further highlighted genes that could potentially serve as therapeutic targets. Furthermore, tNEPC-related methylation signals from this model were detectable in circulating cell free DNA (cfDNA) from mCRPC patients undergoing androgen-targeting therapies and were associated with a faster time to clinical progression. These potential biomarkers could help with identifying patients with aggressive disease.
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http://dx.doi.org/10.1038/s41598-021-85812-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988053PMC
March 2021

The transcriptional landscape of Shh medulloblastoma.

Nat Commun 2021 03 19;12(1):1749. Epub 2021 Mar 19.

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

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.
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http://dx.doi.org/10.1038/s41467-021-21883-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979819PMC
March 2021

netDx: Software for building interpretable patient classifiers by multi-'omic data integration using patient similarity networks.

F1000Res 2020 15;9:1239. Epub 2020 Oct 15.

The Donnelly Centre, University of Toronto, Toronto, Canada.

Patient classification based on clinical and genomic data will further the goal of precision medicine. Interpretability is of particular relevance for models based on genomic data, where sample sizes are relatively small (in the hundreds), increasing overfitting risk netDx is a machine learning method to integrate multi-modal patient data and build a patient classifier. Patient data are converted into networks of patient similarity, which is intuitive to clinicians who also use patient similarity for medical diagnosis. Features passing selection are integrated, and new patients are assigned to the class with the greatest profile similarity. netDx has excellent performance, outperforming most machine-learning methods in binary cancer survival prediction. It handles missing data - a common problem in real-world data - without requiring imputation. netDx also has excellent interpretability, with native support to group genes into pathways for mechanistic insight into predictive features. The netDx Bioconductor package provides multiple workflows for users to build custom patient classifiers. It provides turnkey functions for one-step predictor generation from multi-modal data, including feature selection over multiple train/test data splits. Workflows offer versatility with custom feature design, choice of similarity metric; speed is improved by parallel execution. Built-in functions and examples allow users to compute model performance metrics such as AUROC, AUPR, and accuracy. netDx uses RCy3 to visualize top-scoring pathways and the final integrated patient network in Cytoscape. Advanced users can build more complex predictor designs with functional building blocks used in the default design. Finally, the netDx Bioconductor package provides a novel workflow for pathway-based patient classification from sparse genetic data.
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http://dx.doi.org/10.12688/f1000research.26429.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883323PMC
April 2021

Biological and therapeutic implications of a unique subtype of NPM1 mutated AML.

Nat Commun 2021 02 16;12(1):1054. Epub 2021 Feb 16.

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

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.
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http://dx.doi.org/10.1038/s41467-021-21233-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886883PMC
February 2021

PRMT5 inhibition disrupts splicing and stemness in glioblastoma.

Nat Commun 2021 02 12;12(1):979. Epub 2021 Feb 12.

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

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.
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http://dx.doi.org/10.1038/s41467-021-21204-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881162PMC
February 2021

Sphingosine-1-phosphate receptor 3 potentiates inflammatory programs in normal and leukemia stem cells to promote differentiation.

Blood Cancer Discov 2021 Jan 1;2(1):32-53. Epub 2020 Dec 1.

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

Acute myeloid leukemia (AML) is a caricature of normal hematopoiesis, driven from leukemia stem cells (LSC) that share some hematopoietic stem cell (HSC) programs including responsiveness to inflammatory signaling. Although inflammation dysregulates mature myeloid cells and influences stemness programs and lineage determination in HSC by activating stress myelopoiesis, such roles in LSC are poorly understood. Here, we show that S1PR3, a receptor for the bioactive lipid sphingosine-1-phosphate, is a central regulator which drives myeloid differentiation and activates inflammatory programs in both HSC and LSC. S1PR3-mediated inflammatory signatures varied in a continuum from primitive to mature myeloid states across AML patient cohorts, each with distinct phenotypic and clinical properties. S1PR3 was high in LSC and blasts of mature myeloid samples with linkages to chemosensitivity, while S1PR3 activation in primitive samples promoted LSC differentiation leading to eradication. Our studies open new avenues for therapeutic target identification specific for each AML subset.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116590PMC
January 2021

Large-scale survey and database of high affinity ligands for peptide recognition modules.

Mol Syst Biol 2020 12;16(12):e9310

The Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Many proteins involved in signal transduction contain peptide recognition modules (PRMs) that recognize short linear motifs (SLiMs) within their interaction partners. Here, we used large-scale peptide-phage display methods to derive optimal ligands for 163 unique PRMs representing 79 distinct structural families. We combined the new data with previous data that we collected for the large SH3, PDZ, and WW domain families to assemble a database containing 7,984 unique peptide ligands for 500 PRMs representing 82 structural families. For 74 PRMs, we acquired enough new data to map the specificity profiles in detail and derived position weight matrices and binding specificity logos based on multiple peptide ligands. These analyses showed that optimal peptide ligands resembled peptides observed in existing structures of PRM-ligand complexes, indicating that a large majority of the phage-derived peptides are likely to target natural peptide-binding sites and could thus act as inhibitors of natural protein-protein interactions. The complete dataset has been assembled in an online database (http://www.prm-db.org) that will enable many structural, functional, and biological studies of PRMs and SLiMs.
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http://dx.doi.org/10.15252/msb.20199310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724964PMC
December 2020

Tempora: Cell trajectory inference using time-series single-cell RNA sequencing data.

PLoS Comput Biol 2020 09 9;16(9):e1008205. Epub 2020 Sep 9.

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

Single-cell RNA sequencing (scRNA-seq) can map cell types, states and transitions during dynamic biological processes such as tissue development and regeneration. Many trajectory inference methods have been developed to order cells by their progression through a dynamic process. However, when time series data is available, most of these methods do not consider the available time information when ordering cells and are instead designed to work only on a single scRNA-seq data snapshot. We present Tempora, a novel cell trajectory inference method that orders cells using time information from time-series scRNA-seq data. In performance comparison tests, Tempora inferred known developmental lineages from three diverse tissue development time series data sets, beating state of the art methods in accuracy and speed. Tempora works at the level of cell clusters (types) and uses biological pathway information to help identify cell type relationships. This approach increases gene expression signal from single cells, processing speed, and interpretability of the inferred trajectory. Our results demonstrate the utility of a combination of time and pathway information to supervise trajectory inference for scRNA-seq based analysis.
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http://dx.doi.org/10.1371/journal.pcbi.1008205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505465PMC
September 2020

Dynamics of the cell-free DNA methylome of metastatic prostate cancer during androgen-targeting treatment.

Epigenomics 2020 08 1;12(15):1317-1332. Epub 2020 Sep 1.

Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, ON, M5G 1X5, Canada.

We examined methylation changes in cell-free DNA (cfDNA) in metastatic castration-resistant prostate cancer (mCRPC) during treatment. Genome-wide methylation analysis of sequentially collected cfDNA samples derived from mCRPC patients undergoing androgen-targeting therapy was performed. Alterations in methylation states of genes previously implicated in prostate cancer progression were observed and patients that maintained methylation changes throughout therapy tended to have a longer time to clinical progression. Importantly, we also report that markers associated with a highly aggressive form of the disease, neuroendocrine-CRPC, were associated with a faster time to clinical progression. Our findings highlight the potential of monitoring the cfDNA methylome during therapy in mCRPC, which may serve as predictive markers of response to androgen-targeting agents.
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http://dx.doi.org/10.2217/epi-2020-0173DOI Listing
August 2020

Wnt activation as a therapeutic strategy in medulloblastoma.

Nat Commun 2020 08 28;11(1):4323. Epub 2020 Aug 28.

McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, L8S 4K1, Canada.

Medulloblastoma (MB) is defined by four molecular subgroups (Wnt, Shh, Group 3, Group 4) with Wnt MB having the most favorable prognosis. Since prior reports have illustrated the antitumorigenic role of Wnt activation in Shh MB, we aimed to assess the effects of activated canonical Wnt signaling in Group 3 and 4 MBs. By using primary patient-derived MB brain tumor-initiating cell (BTIC) lines, we characterize differences in the tumor-initiating capacity of Wnt, Group 3, and Group 4 MB. With single cell RNA-seq technology, we demonstrate the presence of rare Wnt-active cells in non-Wnt MBs, which functionally retain the impaired tumorigenic potential of Wnt MB. In treating MB xenografts with a Wnt agonist, we provide a rational therapeutic option in which the protective effects of Wnt-driven MBs may be augmented in Group 3 and 4 MB and thereby support emerging data for a context-dependent tumor suppressive role for Wnt/β-catenin signaling.
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http://dx.doi.org/10.1038/s41467-020-17953-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455709PMC
August 2020

Single-Cell Profiling Shows Murine Forebrain Neural Stem Cells Reacquire a Developmental State when Activated for Adult Neurogenesis.

Cell Rep 2020 08;32(6):108022

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:

The transitions from developing to adult quiescent and activated neural stem cells (NSCs) are not well understood. Here, we use single-cell transcriptional profiling and lineage tracing to characterize these transitions in the murine forebrain. We show that the two forebrain NSC parental populations, embryonic cortex and ganglionic eminence radial precursors (RPs), are highly similar even though they make glutamatergic versus gabaergic neurons. Both RP populations progress linearly to transition from a highly active embryonic to a dormant adult stem cell state that still shares many similarities with embryonic RPs. When adult NSCs of either embryonic origin become reactivated to make gabaergic neurons, they acquire a developing ganglionic eminence RP-like identity. Thus, transitions from embryonic RPs to adult NSCs and back to neuronal progenitors do not involve fundamental changes in cell identity, but rather reflect conversions between activated and dormant NSC states that may be determined by the niche environment.
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http://dx.doi.org/10.1016/j.celrep.2020.108022DOI Listing
August 2020

Meta-analysis of gene expression profiles of lean and obese PCOS to identify differentially regulated pathways and risk of comorbidities.

Comput Struct Biotechnol J 2020 21;18:1735-1745. Epub 2020 Jun 21.

The Donnelly Centre, University of Toronto, Toronto, ON, Canada.

Polycystic ovary syndrome (PCOS) is a complex multigenic disorder and women with PCOS suffer from several comorbidities. Although, obesity is a known risk factor for PCOS, the incidence of lean women with PCOS is on the rise. A systematic and comparative study on lean and obese PCOS with respect to genes, pathways and comorbidity analysis has not been attempted so far. Analysis of differentially expressed genes (DEGs) across tissue types for lean and obese PCOS revealed that the majority of them were downregulated for lean and obese PCOS. Ovarian and endometrial tissues shared several commonly dysregulated genes, suggesting shared PCOS pathophysiology mechanisms exist across tissues. Several pathways for cellular homeostasis, such as inflammation and immune response, insulin signaling, steroidogenesis, hormonal and metabolic signaling, regulation of gonadotrophic hormone secretion, cell structure and signaling that are known to be affected in PCOS were found to be enriched in our gene expression analysis of lean and obese PCOS. The gene-disease network is denser for obese PCOS with a higher comorbidity score as compared to lean PCOS.
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http://dx.doi.org/10.1016/j.csbj.2020.06.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352056PMC
June 2020

Generation of Functional Liver Sinusoidal Endothelial Cells from Human Pluripotent Stem-Cell-Derived Venous Angioblasts.

Cell Stem Cell 2020 08 7;27(2):254-269.e9. Epub 2020 Jul 7.

McEwen Stem Cell Institute, University Health Network, Toronto, ON M5G1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G1L7, Canada. Electronic address:

Liver sinusoidal endothelial cells (LSECs) form a highly specialized microvasculature that plays a critical role in liver function and disease. To better understand this role, we developed a strategy to generate LSECs from human pluripotent stem cells (hPSCs) by first optimizing the specification of arterial and venous angioblasts and derivative endothelial populations. Induction of a LSEC-like fate by hypoxia, cyclic AMP (cAMP) agonism, and transforming growth factor β (TGF-β) inhibition revealed that venous endothelial cells responded more rapidly and robustly than the arterial cells to upregulate LSEC markers and functions in vitro. Upon intrahepatic transplantation in neonates, venous angioblasts engrafted the liver and generated mature, fenestrated LSECs with scavenger functions and molecular profiles of primary human LSECs. When transplanted into the liver of adult mice, angioblasts efficiently gave rise to mature LSECs with robust factor VIII (FVIII) production. Humanization of the murine liver with hPSC-derived LSECs provides a tractable system for studying the biology of this key liver cell type.
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http://dx.doi.org/10.1016/j.stem.2020.06.007DOI Listing
August 2020

Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21.

J Exp Med 2020 08;217(8)

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.
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http://dx.doi.org/10.1084/jem.20191094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398174PMC
August 2020

Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal.

Cell Stem Cell 2020 06 15;26(6):926-937.e10. Epub 2020 May 15.

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

Leukemic stem cells (LSCs) rely on oxidative metabolism and are differentially sensitive to targeting mitochondrial pathways, which spares normal hematopoietic cells. A subset of mitochondrial proteins is folded in the intermembrane space via the mitochondrial intermembrane assembly (MIA) pathway. We found increased mRNA expression of MIA pathway substrates in acute myeloid leukemia (AML) stem cells. Therefore, we evaluated the effects of inhibiting this pathway in AML. Genetic and chemical inhibition of ALR reduces AML growth and viability, disrupts LSC self-renewal, and induces their differentiation. ALR inhibition preferentially decreases its substrate COX17, a mitochondrial copper chaperone, and knockdown of COX17 phenocopies ALR loss. Inhibiting ALR and COX17 increases mitochondrial copper levels which in turn inhibit S-adenosylhomocysteine hydrolase (SAHH) and lower levels of S-adenosylmethionine (SAM), DNA methylation, and chromatin accessibility to lower LSC viability. These results provide insight into mechanisms through which mitochondrial copper controls epigenetic status and viability of LSCs.
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http://dx.doi.org/10.1016/j.stem.2020.04.010DOI Listing
June 2020

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain.

Cell Syst 2020 04;10(4):333-350.e14

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Broad Institute of Massachusetts Institute of Technology and Harvard University, Boston, MA, USA.

Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.
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http://dx.doi.org/10.1016/j.cels.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938770PMC
April 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

A reference map of the human binary protein interactome.

Nature 2020 04 8;580(7803):402-408. Epub 2020 Apr 8.

Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome, transcriptome and proteome data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.
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http://dx.doi.org/10.1038/s41586-020-2188-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7169983PMC
April 2020

The mitochondrial peptidase, neurolysin, regulates respiratory chain supercomplex formation and is necessary for AML viability.

Sci Transl Med 2020 04;12(538)

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

Neurolysin (NLN) is a zinc metallopeptidase whose mitochondrial function is unclear. We found that NLN was overexpressed in almost half of patients with acute myeloid leukemia (AML), and inhibition of NLN was selectively cytotoxic to AML cells and stem cells while sparing normal hematopoietic cells. Mechanistically, NLN interacted with the mitochondrial respiratory chain. Genetic and chemical inhibition of NLN impaired oxidative metabolism and disrupted the formation of respiratory chain supercomplexes (RCS). Furthermore, NLN interacted with the known RCS regulator, LETM1, and inhibition of NLN disrupted LETM1 complex formation. RCS were increased in patients with AML and positively correlated with NLN expression. These findings demonstrate that inhibiting RCS formation selectively targets AML cells and stem cells and highlights the therapeutic potential of pharmacologically targeting NLN in AML.
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http://dx.doi.org/10.1126/scitranslmed.aaz8264DOI Listing
April 2020

Nanoparticle Uptake in a Spontaneous and Immunocompetent Woodchuck Liver Cancer Model.

ACS Nano 2020 04 14;14(4):4698-4715. Epub 2020 Apr 14.

Soham and Shaila Ajmera Family Transplant Centre, Toronto General Research Institute, University Health Network, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4.

There is a tremendous focus on the application of nanomaterials for the treatment of cancer. Nonprimate models are conventionally used to assess the biomedical utility of nanomaterials. However, these animals often lack an intact immunological background, and the tumors in these animals do not develop spontaneously. We introduce a preclinical woodchuck hepatitis virus-induced liver cancer model as a platform for nanoparticle (NP)-based experiments. Liver cancer development in these out-bred animals occurs as a result of persistent viral infection, mimicking human hepatitis B virus-induced HCC development. We highlight how this model addresses key gaps associated with other commonly used tumor models. We employed this model to (1) track organ biodistribution of gold NPs after intravenous administration, (2) examine their subcellular localization in the liver, (3) determine clearance kinetics, and (4) characterize the identity of hepatic macrophages that take up NPs using RNA-sequencing (RNA-seq). We found that the liver and spleen were the primary sites of NP accumulation. Subcellular analyses revealed accumulation of NPs in the lysosomes of CD14 cells. Through RNA-seq, we uncovered that immunosuppressive macrophages within the woodchuck liver are the major cell type that take up injected NPs. The woodchuck-HCC model has the potential to be an invaluable tool to examine NP-based immune modifiers that promote host anti-tumor immunity.
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http://dx.doi.org/10.1021/acsnano.0c00468DOI Listing
April 2020

Potential therapeutic targets for lung repair during human lung perfusion.

Eur Respir J 2020 04 9;55(4). Epub 2020 Apr 9.

Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, ON, Canada

Introduction: The lung perfusion (EVLP) technique has been developed to assess the function of marginal donor lungs and has significantly increased donor lung utilisation. EVLP has also been explored as a platform for donor lung repair through injury-specific treatments such as antibiotics or fibrinolytics. We hypothesised that actively expressed pathways shared between transplantation and EVLP may reveal common mechanisms of injury and potential therapeutic targets for lung repair prior to transplantation.

Materials And Methods: Retrospective transcriptomics analyses were performed with peripheral tissue biopsies from "donation after brain death" lungs, with 46 pre-/post-transplant pairs and 49 pre-/post-EVLP pairs. Pathway analysis was used to identify and compare the responses of donor lungs to transplantation and to EVLP.

Results: 22 pathways were enriched predominantly in transplantation, including upregulation of lymphocyte activation and cell death and downregulation of metabolism. Eight pathways were enriched predominantly in EVLP, including downregulation of leukocyte functions and upregulation of vascular processes. 27 pathways were commonly enriched, including activation of innate inflammation, cell death, heat stress and downregulation of metabolism and protein synthesis. Of the inflammatory clusters, Toll-like receptor/innate immune signal transduction adaptor signalling had the greatest number of nodes and was central to inflammation. These mechanisms have been previously speculated as major mechanisms of acute lung injury in animal models.

Conclusion: EVLP and transplantation share common molecular features of injury including innate inflammation and cell death. Blocking these pathways during EVLP may allow for lung repair prior to transplantation.
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http://dx.doi.org/10.1183/13993003.02222-2019DOI Listing
April 2020
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