Notable findings and advances from the past six months
by DANIEL PAUL PEREZ
Chief Science Officer, FSH Society
Asterisk denotes FSH Society funding acknowledged in paper.
“Conservation and innovation in the DUX4-family gene network,” from the laboratory of Stephen J. Tapscott at the Fred Hutchinson Cancer Center in Seattle, Washington (Whiddon et al. Nat Genet. 2017 Jun;49(6):935-940. doi: 10.1038/ng.3846. Epub 2017 May 1).
“Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons,” from the laboratory of Bradley R. Cairns at the University of Utah (Hendrickson et al. Nat Genet. 2017 Jun;49(6):925-934).
“DUX-family transcription factors regulate zygotic genome activation in placental mammals,” from the laboratory of Didier Trono at École Polytechnique Fédérale de Lausanne (EPFL), Switzerland (De Iaco et al. Nat Genet. 2017 Jun;49(6):941-945).
This trio of papers in the same issue of Nature Genetics identifies the master role played by DUX4 in early embryogenesis. This body of work is absolutely key and begins to unwrap the normal biological function of DUX4, which is very important to understand if we are to attempt to treat FSHD by suppressing the unwanted negative effects of DUX4.
* “Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FacioScapuloHumeral Muscular Dystrophy (FSHD),” from the laboratory of Alexandra Belayew and Eugénie Ansseau at the University of Mons, Belgium (Ansseau et al. Genes (Basel). 2017 Mar 3;8(3). pii: E93. doi: 10.3390/genes8030093).
DUX4 suppression is a promising therapy for individuals with FSHD. This paper reviews what is known about DUX4 mRNAs observed in muscle and stem cells, and the use of antisense oligonucleotides (AOs) against them. This paper introduces the unique idea that DUX4c, a molecule closely similar to DUX4, might be another therapeutic avenue in FSHD. DUX4 suppression therapy may be challenging because of the working assumption that FSHD is caused by a burst-like feature of DUX4 expression, with a low number of muscle nuclei expressing abundant amounts of DUX4. This implies that AOs might need to be administered in large and repeated doses to achieve a therapeutic effect and may cause significant side effects.
* “DUX4-induced dsRNA and MYC mRNA stabilization activate apoptotic pathways in human cell models of facioscapulohumeral dystrophy,” from the laboratory of Stephen J. Tapscott at the Fred Hutchinson Cancer Center in Seattle, Washington (Shadle et al. PLoS Genet. 2017 Mar 8;13(3):e1006658. doi: 10.1371/journal.pgen.1006658. eCollection 2017 Mar).
This work reports a new mechanism of DUX4 toxicity mediated by double-stranded RNA and helps us understand why we see a strong immune response when DUX4 is expressed—another important facet in understanding DUX4’s toxicity in the muscle. This adds to other findings that endogenous DUX4 expression affects RNA metabolism and cell signaling pathways, inhibits protein turnover, and promotes aggregation of proteins such as TDP-43 (akin to similar muscle and nerve diseases).
“MRI as outcome measure in facioscapulohumeral muscular dystrophy: 1-year follow-up of 45 patients,” from the laboratory of John Vissing at the University of Copenhagen, Denmark (Anderson et al. J Neurol. 2017 Mar;264(3):438-447. doi: 10.1007/s00415-016-8361-3. Epub 2016 Dec 20).
This paper expands on previous work on magnetic resonance imaging (MRI) to evaluate the extent of pathology in FSHD and the different phases of disease at the level of single muscles. This study “shows that MRI provides an objective measure of disease progression, often before changes can be appreciated in strength and functional tests.” The development of imaging and tissue biomarkers is of major importance for measuring changes in disease progression and for clinical trials.
* “Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity,” from the laboratory of Fabiola Moretti of the Institute of Cell Biology and Neurobiology, National Research Council of Italy, Rome (Teveroni et al. J Clin Invest. 2017 Apr 3;127(4):1531-1545. doi: 10.1172/JCI89401).
This seminal work identifies estrogens as a potential disease modifier that underlies sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease. Results demonstrate that estrogens do so without affecting cell proliferation or survival. This may help with designing novel hormone-based therapies for FSHD. In addition, this paper provides evidence of nuclear localization of DUX4 during early muscle differentiation, which perhaps could serve as an additional factor or modifier that can be used to help interfere with DUX4 activity and related toxicity.
* “Muscle Microdialysis to Investigate Inflammatory Biomarkers in Facioscapulohumeral Muscular Dystrophy,” from the laboratory of Enzo Ricci at the Università Cattolica del Sacro Cuore, Rome, Italy (Tasca et al. Mol Neurobiol. 2017 Apr 29. doi: 10.1007/s12035-017-0563-x).
This pioneering clinical study combines magnetic resonance imaging (MRI) with microdialysis, a procedure performed by inserting a very thin capillary tube into a muscle for a period of time, giving continuous sampling of the interstitial fluid. Analysis of the molecules in the microdialysis samples has identified inflammatory cytokines that correlate with inflammatory features seen by MRI in muscles in different disease stages in FSHD individuals. The goal is to establish molecular biomarkers and imaging markers of early muscle damage and thus acquire indicators to monitor early disease activity in FSHD.
“A distal auxiliary element facilitates cleavage and polyadenylation of Dux4 mRNA in the pathogenic haplotype of FSHD,” from the laboratory of Eric Wagner at the University of Texas Medical Branch at Galveston (Peart and Wagner. Hum Genet. 2017 May 24. doi: 10.1007/s00439-017-1813-8).
This interesting paper identifies a novel element that is important for DUX4 mRNA processing and might be a new target for antisense oligonucleotide-based therapeutics.
“SMCHD1 regulates a limited set of gene clusters on autosomal chromosomes,” from the laboratory of Silvère van der Maarel at the University of Leiden, the Netherlands (Mason et al. Skelet Muscle. 2017 Jun 6;7(1):12. doi: 10.1186/s13395-017-0129-7).
This paper is the first to investigate the genomewide methylation effects resulting from mutations in SMCHD1, the gene that causes FSHD2 and is a “modifier” that makes FSHD1 worse. The work expands the idea of FSHD as an epigenetic or heterochromatin abnormality disorder in which genomewide changes of heterochromatin and SMCHD1 function predispose to or initiate FSHD.
“Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures,” from the laboratory of Daniel G. Miller at the University of Washington, Seattle (Haynes et al. Skelet Muscle. 2017 Jun 21;7(1):13. doi: 10.1186/s13395-017-0130-1).
This work finds that DUX4, PAX3, and PAX7 have distinct spatial patterns of expression during differentiation of induced pluripotent stem cells (iPSCs) derived from FSHD patients. The study investigates how DUX4, PAX3, or PAX7 functions interact and compete as the iPSCs mature into muscle-like cells. The study indicates that these three transcription factors are “unlikely to compete for the same genomic binding sites in differentiating stem cell cultures.” Understanding these dynamics would be important for designing therapies based on cell transplantation.
* “BET bromodomain inhibitors and agonists of the beta-2 adrenergic receptor identified in screens for compounds that inhibit DUX4 expression in FSHD muscle cells,” from the laboratory of Fran Sverdrup at St. Louis University, Missouri (Campbell et al. Skelet Muscle. 2017 Sep 4;7(1):16. doi: 10.1186/s13395-017-0134-x).
A prime avenue for treating FSHD is to clamp down on DUX4 protein in skeletal muscle. This study screened collections of drugs with epigenetic activities (which regulate gene expression) that are being used or tested in human patients. These efforts have uncovered possibilities to use compounds involved with bromodomain and extra-terminal (BET) proteins and beta adrenergic signaling for clinical trials.
* “An Instrumented Timed Up and Go in Facioscapulohumeral Muscular Dystrophy,” from the laboratory of Jeffrey Statland at the University of Kansas Medical Center, Kansas City (Huisinga et al. Muscle Nerve. 2017 Sep 6. doi: 10.1002/mus.25955).
This groundbreaking paper adds tools to modernize and fine-tune methods to capture natural history data (needed to run clinical trials). Using wireless motion-capture systems, this study shows the “instrumented timed up and go” task (in which a seated patient stands up and walks a distance) is reliable, abnormal in FSHD, and could distinguish among patients with differing degrees of severity of FSHD symptoms.
“Muscle pathology from stochastic low level DUX4 expression in an FSHD mouse model,” from the laboratory of Michael Kyba at the University of Minnesota in Minneapolis (Bosnakovski et al. Nat Commun. 2017 Sep 15;8(1):550. doi: 10.1038/s41467-017-00730-1).
This paper is significant in providing a new in vivo (living animal) model system for FSHD—something the field really needs for clinical trials moving forward. (See our story on this paper.)
“The DUX4 homeodomains mediate inhibition of myogenesis and are functionally exchangeable with the Pax7 homeodomain,” from the laboratory of Michael Kyba at the University of Minnesota in Minneapolis (Bosnakovski et al. J Cell Sci. 2017 Sep 21. pii: jcs.205427. doi: 10.1242/jcs.205427).
This insightful paper examines the Pax3, Pax7, and DUX4 interaction and relevance to the myogenic lineage, including postnatal cells. It sets a framework to ask if key toxicity-related targets have a motif recognized by DUX4, Pax3, and Pax7, or possibly whether homeodomains (the part of the protein that attaches to specific regulatory regions of the target genes) are competing for a homeodomain-interacting protein. If so, Pax3 or Pax7 could deplete the DUX4 complex of a key cofactor necessary for full DUX4 activity. This refined understanding of DUX4 processing/mechanisms and toxic activity will yield new avenues for therapies.
* “p53-independent DUX4 pathology in cell and animal models of facioscapulohumeral muscular dystrophy,” from the laboratory of Michael Kyba at the University of Minnesota in Minneapolis (Bosnakovski et al. Dis Model Mech. 2017 Oct 1;10(10):1211-1216. doi: 10.1242/dmm.030064. Epub 2017 Jul 28).
Studies in mice have shown that toxicity caused by overexpressing DUX4 can disturb myogenic (muscle development) pathways, potentially by competing with PAX3 and PAX7, both regulators of myogenesis. In addition, cell death (apoptosis) brought on by DUX4 is thought to be dependent on its ability to bind DNA, possibly through the p53 pathway. This paper shows that DUX4 expression does not induce p53 but only its target gene Cdkn1a in a p53-independent manner. This finding is important for understanding which specific points along a pathway are potential targets for therapies.