P38a Regulates Expression of DUX4 in a Model of Facioscapulohumeral Muscular Dystrophy S

Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2020/06/23/jpet.119.264689.DC1 1521-0103/374/3/489–498$35.00 https://doi.org/10.1124/jpet.119.264689 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 374:489–498, September 2020 Copyright ª 2020 The Author(s) This is an open access article distributed under the CC BY Attribution 4.0 International license. p38a Regulates Expression of DUX4 in a Model of Facioscapulohumeral Muscular Dystrophy s L. Alejandro Rojas, Erin Valentine, Anthony Accorsi, Joseph Maglio, Ning Shen, Alan Robertson, Steven Kazmirski, Peter Rahl, Rabi Tawil, Diego Cadavid, Lorin A. Thompson, Lucienne Ronco, Aaron N. Chang, Angela M. Cacace, and Owen Wallace Fulcrum Therapeutics, Cambridge, Massachusetts (L.A.R., E.V., A.A., J.M., N.S., A.R., S.K., P.R., D.C., L.A.T., L.R., A.N.C., A.M.C., O.W.) and University of Rochester Medical Center, Department of Neurology, Rochester, New York (R.T.) Received December 16, 2019; accepted May 26, 2020 Downloaded from ABSTRACT Facioscapulohumeral muscular dystrophy (FSHD) is caused by of which are DUX4 target genes. Our results reveal a novel and the loss of repression at the D4Z4 locus leading to aberrant double apparently critical role for p38a in the aberrant activation of DUX4 homeobox 4 (DUX4) expression in skeletal muscle. Activation of in FSHD and support the potential of p38a/b inhibitors as effective jpet.aspetjournals.org this early embryonic transcription factor results in the expression therapeutics to treat FSHD at its root cause. of its target genes causing muscle fiber death. Although progress toward understanding the signals driving DUX4 expression has SIGNIFICANCE STATEMENT been made, the factors and pathways involved in the transcrip- Using patient-derived facioscapulohumeral muscular dystrophy tional activation of this gene remain largely unknown. Here, we (FSHD) myotubes, we characterize the pharmacological relation- describe the identification and characterization of p38a as a novel ships between p38a/b inhibition, double homeobox 4 (DUX4) regulator of DUX4 expression in FSHD myotubes. By using expression, its downstream transcriptional program, and muscle multiple highly characterized, potent, and specific inhibitors of cell death. p38a/b inhibition results in potent and specific DUX4 at ASPET Journals on September 27, 2021 p38a/b, we show a robust reduction of DUX4 expression, activity, downregulation across multiple genotypes without significant and cell death across patient-derived FSHD1 and FSHD2 lines. effects in the process of myogenesis in vitro. These findings RNA-seq profiling reveals that a small number of genes are highlight the potential of p38a/b inhibitors for the treatment of differentially expressed upon p38a/b inhibition, the vast majority FSHD, a condition that today has no approved therapies. Introduction FSHD is caused by aberrant expression of the double homeobox 4 (DUX4) gene, a homeobox transcription factor in Facioscapulohumeral muscular dystrophy (FSHD) is a rare the skeletal muscle of patients. This gene is located within the and disabling condition with an estimated worldwide popula- D4Z4 macrosatellite repeats on chromosome 4q35. DUX4 is tion prevalence of between 1 in 8000 and 20,000 (Deenen et al., not expressed in adult skeletal muscle when the number of 2014; Statland and Tawil, 2014). Most cases are familial and repeat units (RUs) is .10, and the locus is properly silenced inherited in an autosomal dominant fashion, and about 30% of (Lemmers et al., 2010). In most patients with FSHD (FSHD1), cases are known to be sporadic. FSHD is characterized by the D4Z4 array is contracted to 1–9 RUs in one allele. Loss of progressive skeletal muscle weakness affecting the face, should- these repetitive elements leads to derepression of the D4Z4 ers, arms, and trunk, which is followed by weakness of the distal locus and ensuing aberrant DUX4 expression in skeletal lower extremities and pelvic girdle (Tawil et al., 2015). There are muscle (de Greef et al., 2009; Wang et al., 2019). In FSHD2, currently no approved treatments for this condition. patients manifest similar signs and symptoms as described above but genetically differ from FSHD1. These patients have Financial disclosure: L.A.R., E.V., A.A., J.M., N.S., A.R., S.K., P.R., D.C., longer D4Z4 arrays but exhibit similar derepression of the L.A.T., L.R., A.N.C., A.M.C., and O.W. are or have been employees and stockholders at Fulcrum Therapeutics. locus caused by mutations in SMCHD1, an important factor in An earlier version of this paper appears in bioRxiv under the DOI: https:// the proper deposition of DNA methylation across the genome doi.org/10.1101/700195. https://doi.org/10.1124/jpet.119.264689. (Jones et al., 2014, 2015; Calandra et al., 2016; Jansz et al., s This article has supplemental material available at jpet.aspetjournals.org. 2017; Dion et al., 2019). Similarly, modifiers of the disease, ABBREVIATIONS: abs(FC), absolute fold change; BET, bromodomain and extraterminal motif protein; DNMT3B, DNA methyltransferase 3B; DUX4, double homeobox 4; FDR, false discovery rate; FSHD, facioscapulohumeral muscular dystrophy; HMBS, hydroxymethylbilane synthase; HSP27, heat shock protein 27; MAPK, mitogen-activated protein kinase; MBD, methyl-CpG binding protein-like; MHC, myosin heavy chain; MSD, Mesoscale Diagnostics; MYOG, myogenin; POLR2A, RNA polymerase II subunit A; qPCR, quantitative polymerase chain reaction; RT, reverse transcription; RU, repeat unit; siRNA, small interfering RNA; SLC34A2, solute carrier 34A2; SMCHD1, structural maintenance of chromosomes flexible hinge domain containing 1; TRIM, tripartite motif–containing. 489 490 Rojas et al. such as DNMT3B, are thought to also participate in the program, and cell death in FSHD myotubes across a vari- establishment of silencing of this region (van den Boogaard ety of FSHD1 and FSHD2 genotypes. Using RNA-seq and et al., 2016b). high-content image analysis, we also demonstrated that myo- DUX4 expression in skeletal muscle as a result of the D4Z4 genesis is not affected at concentrations that result in down- repeat contraction or SMCHD1 mutations leads to activation of regulation of DUX4. a downstream transcriptional program that causes FSHD (Yao et al., 2014; Jagannathan et al., 2016; Shadle et al., 2017). Major target genes of DUX4 are members of the DUX family itself and Materials and Methods other homeobox transcription factors. Additional target genes Cell Lines and Cell Culture. Immortalized myoblasts from include highly homologous gene families, including the prefer- FSHD (AB1080FSHD26 C6) and healthy individuals (AB1167C20FL) entially expressed in melanoma family (PRAMEF), tripartite were generated and obtained from the Institut Myologie, France. In motif–containing (TRIM), and methyl-CpG–binding protein-like short, primary myoblast cultures were obtained from patient samples (MBD3L) (Geng et al., 2012; Tawil et al., 2014; Yao et al., 2014; and immortalized by overexpression of telomerase reverse transcrip- Shadle et al., 2017). Expression of DUX4 and its downstream tase (TERT) and cyclin-dependent kinase 4 (CDK4) (Krom et al., transcriptional program in skeletal muscle cells is toxic, leading 2012). Primary myoblasts were isolated from FSHD muscle biopsies to dysregulation of multiple pathways and resulting in impair- and were obtained from University of Rochester. Immortalized myoblasts were expanded on gelatin-coated dishes ment of contractile function and cell death (Bosnakovski et al., (ES-006-B; EMD Millipore) using skeletal muscle cell growth media Downloaded from 2014; Tawil et al., 2014; Himeda et al., 2015; Homma et al., 2015; (C-23060; Promocell) supplemented with 15% FBS (16000044; Ther- Rickard et al., 2015; Statland et al., 2015). moFisher). Primary myoblasts were also expanded on gelatin-coated Several groups have made progress toward understanding plates but using media containing Ham’s F-10 Nutrient Mix the molecular mechanisms regulating DUX4 expression (van (11550043; ThermoFisher), 20% FBS, and 0.5% chicken embryo den Boogaard et al., 2016a,b; Campbell et al., 2017, 2018; Cruz extract (100-163P; Gemini Bio-product). For differentiation, immor- et al., 2018; Oliva et al., 2019). However, factors that drive talized or primary myoblasts were grown to confluency in matrigel- transcriptional activation of DUX4 in patients with FSHD are coated plates (356234; Corning), and growth media was exchanged for jpet.aspetjournals.org still largely unknown. By screening our annotated chemical differentiation media (Nb4-500; Brainbits) after a PBS wash. DMSO probe library to identify disease-modifying small-molecule (vehicle) or compounds (previously dissolved in DMSO at 10-mM stock concentrations) were added at the desired concentration at the time drug targets that reduce DUX4 expression in FSHD myo- differentiation media was exchanged and maintained in the plates tubes, we have identified multiple chemical scaffolds that until harvesting or analysis. inhibit p38a and b mitogen-activated protein kinase (MAPK). Small-Molecule Compounds and Antisense Oligonucleoti- We found that inhibitors of p38a kinase or its genetic des. SB239063, Pamapimod, LY2228820, and Losmapimod were knockdown reduce DUX4 and its downstream gene expression purchased from Selleck Chem (S7741, S8125, S1494, and S7215). at ASPET Journals on September 27, 2021 program in FSHD myotubes, thereby impacting the core Ten-millimolar stock solutions in DMSO were

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