Publications by authors named "Fadi J Najm"

10 Publications

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Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Cell 2020 04 3;181(2):382-395.e21. Epub 2020 Apr 3.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. Electronic address:

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2020.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426147PMC
April 2020

Diverse Chemical Scaffolds Enhance Oligodendrocyte Formation by Inhibiting CYP51, TM7SF2, or EBP.

Cell Chem Biol 2019 04 14;26(4):593-599.e4. Epub 2019 Feb 14.

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

Small molecules that promote oligodendrocyte formation have been identified in "drug repurposing" screens to nominate candidate therapeutics for diseases in which myelin is lost, including multiple sclerosis. We recently reported that many such molecules enhance oligodendrocyte formation not by their canonical targets but by inhibiting a narrow range of enzymes in cholesterol biosynthesis. Here we identify enhancers of oligodendrocyte formation obtained by screening a structurally diverse library of 10,000 small molecules. Identification of the cellular targets of these validated hits revealed a majority inhibited the cholesterol biosynthesis enzymes CYP51, TM7SF2, or EBP. In addition, evaluation of analogs led to identification of CW3388, a potent EBP-inhibiting enhancer of oligodendrocyte formation poised for further optimization.
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http://dx.doi.org/10.1016/j.chembiol.2019.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474808PMC
April 2019

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Nat Biotechnol 2018 02 18;36(2):179-189. Epub 2017 Dec 18.

Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
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http://dx.doi.org/10.1038/nbt.4048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800952PMC
February 2018

Adaptive Chromatin Remodeling Drives Glioblastoma Stem Cell Plasticity and Drug Tolerance.

Cell Stem Cell 2017 02 15;20(2):233-246.e7. Epub 2016 Dec 15.

Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.
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http://dx.doi.org/10.1016/j.stem.2016.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291795PMC
February 2017

Depletion of Olig2 in oligodendrocyte progenitor cells infected by Theiler's murine encephalomyelitis virus.

J Neurovirol 2016 06 2;22(3):336-48. Epub 2015 Dec 2.

Department of Biology, The University of Tampa, Tampa, FL, USA.

Theiler's murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown. Here, we employed pluripotent stem cell technologies to generate pure populations of mouse OPCs to study the temporal and molecular effects of TMEV infection. Global transcriptome analysis of RNA sequencing data revealed that TMEV infection of OPCs caused significant up-regulation of 1926 genes, whereas 1853 genes were significantly down-regulated compared to uninfected cells. Pathway analysis revealed that TMEV disrupted many genes required for OPC growth and maturation. Down-regulation of Olig2, a transcription factor necessary for OPC proliferation, was confirmed by real-time PCR, immunofluorescence microscopy, and western blot analysis. Depletion of Olig2 was not found to be specific to viral strain and did not require expression of the leader (L) protein, which is a multifunctional protein important for persistence, modulation of gene expression, and cell death. These data suggest that direct infection of OPCs by TMEV may inhibit remyelination during the chronic phase of TMEV-induced demyelinating disease.
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http://dx.doi.org/10.1007/s13365-015-0402-7DOI Listing
June 2016

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.

Nature 2015 Jun 20;522(7555):216-20. Epub 2015 Apr 20.

1] Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA [2] Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.
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http://dx.doi.org/10.1038/nature14335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528969PMC
June 2015

Generation and characterization of epiblast stem cells from blastocyst-stage mouse embryos.

Methods Mol Biol 2013 ;1074:1-13

Department of Genetics and Genome Sciences, Case Western Reserve, University School of Medicine, Cleveland, OH, USA.

Mouse epiblast stem cells (EpiSCs) are pluripotent embryonic cells that can be used to interrogate developmental transitions that occur during gastrulation. EpiSCs can also be robustly differentiated into functional somatic and germ cell derivatives making them a useful tool for studying development and regenerative medicine. Typically, mouse EpiSCs are isolated from the early postimplantation epiblast around 5.5 days post coitum (dpc). This chapter describes the methods for isolation of mouse EpiSCs from preimplantation blastocyst-stage mouse embryos (3.5 dpc). This technique enables the routine ability to derive EpiSC lines as it is much less labor intensive than isolation of EpiSCs from the postimplantation epiblast. We also detail relevant assays used to characterize new EpiSC lines and distinguish them from mouse embryonic stem cells.
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http://dx.doi.org/10.1007/978-1-62703-628-3_1DOI Listing
March 2014

Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells.

Nat Biotechnol 2013 May 14;31(5):426-33. Epub 2013 Apr 14.

Department of Genetics and Genome Sciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to induced oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies.
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http://dx.doi.org/10.1038/nbt.2561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678540PMC
May 2013

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells.

Nat Methods 2011 Sep 25;8(11):957-62. Epub 2011 Sep 25.

Department of Genetics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.
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http://dx.doi.org/10.1038/nmeth.1712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400969PMC
September 2011

Isolation of epiblast stem cells from preimplantation mouse embryos.

Cell Stem Cell 2011 Mar;8(3):318-25

Department of Genetics & Center for Stem Cell and Regenerative Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.
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http://dx.doi.org/10.1016/j.stem.2011.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073125PMC
March 2011