P38α Regulates Expression of DUX4 in Facioscapulohumeral Muscular Dystrophy

FSHD is caused by the loss of repression at the D4Z4 locus leading to DUX4 expression in skeletal muscle, activation of its early embryonic transcriptional program and muscle fiber death. While progress toward understanding the signals driving DUX4 expression has been made, the factors and pathways involved in the transcriptional activation of this gene remain largely unknown. Here, we describe the identification and characterization of p38α as a novel regulator of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent and specific inhibitors of p38α/β, we show a robust reduction of DUX4 expression, activity and cell death across FSHD1 and FSHD2 patient-derived lines. RNA-seq profiling reveals that a small number of genes are differentially expressed upon p38α/β inhibition, the vast majority of which are DUX4 target genes. Our results reveal a novel and apparently critical role for p38a in the aberrant activation of DUX4 in FSHD and support the potential of p38α/β inhibitors as effective therapeutics to treat FSHD at its root cause. Graphical Abstract

of DUX4 expression in FSHD myotubes. By using multiple highly characterized, potent and 23 specific inhibitors of p38α/β, we show a robust reduction of DUX4 expression, activity and cell 24 death across FSHD1 and FSHD2 patient-derived lines. RNA-seq profiling reveals that a small 25 number of genes are differentially expressed upon p38α/β inhibition, the vast majority of which 26 are DUX4 target genes. Our results reveal a novel and apparently critical role for p38a in the 27 aberrant activation of DUX4 in FSHD and support the potential of p38α/β inhibitors as effective 28 therapeutics to treat FSHD at its root cause.

INTRODUCTION 30
Facioscapulohumeral muscular dystrophy (FSHD) is a rare and disabling disease with an 31 estimated worldwide population prevalence of between 1 in 8,000-20,000 (Deenen et al., 2014; 32 Statland and Tawil, 2014). Most cases are familial and inherited in an autosomal dominant fashion 33 and about 30% of cases are known to be sporadic. FSHD is characterized by progressive skeletal 34 muscle weakness affecting the face, shoulders, arms, and trunk, followed by weakness of the 35 distal lower extremities and pelvic girdle. Initial symptoms typically appear in the second decade 36 of life but can occur at any age resulting in significant physical disability in later decades (Tawil et 37 al., 2015). There are currently no approved treatments for this disease. is >10 and the locus is properly silenced (Lemmers et al., 2010). In the majority of patients with 42 FSHD (FSHD1), the D4Z4 array is contracted to 1-9 repeat units on one allele. FSHD1 patients 43 carrying a smaller number of repeats (1-3 units) are on average more severely affected than 44 those with a higher number of repeats (8-9) (Tawil et al., 1996). Loss of these repetitive elements 45 (referred to as contraction) leads to de-repression of the D4Z4 locus and ensuing aberrant DUX4 46 expression activation in skeletal muscle (de Greef et al., 2009;Wang et al., 2018). In FSHD2, 47 patients manifest similar signs and symptoms as described above but genetically differ from 48 vehicle or p38α/β inhibitors. Inhibition of the p38 signaling pathway during differentiation did not 180 induce significant transcriptome changes, and resulted in less than 100 differentially expressed 181 genes (abs(FC)>4; FDR<0.001). Around 80% of these differentially expressed genes were known 182 DUX4-regulated transcripts and were all downregulated after p38 α and β inhibition ( Figure 3B). 183 This set of DUX4-regulated genes overlapped significantly with genes upregulated in FSHD 184 patient muscle biopsies (Wang et al., 2018). Moreover, key driver genes of myogenic programs 185 such as MYOG, MEF and PAX genes and markers of differentiation such as myosin subunits and 186 sarcomere proteins were not affected by p38 inhibition ( Figure 3C). to FSHD pathology (Sandri et al., 2001;Statland et al., 2015). To test this hypothesis in vitro, we 195 evaluated the effect of p38α/β inhibition on apoptosis in FSHD myotubes. We used an antibody 196 recognizing caspase-3 cleavage products by immunofluorescence to quantify changes in the 197 activation of programmed cell death. Cleavage of caspase-3 is a major step in the execution of 198 the apoptosis signaling pathway, leading to the final proteolytic steps that result in cell death (Dix 199 Figure 4B). Moreover, we measured SLC34A2, a DUX4 target gene product 205 using a similar immunofluorescence assay ( Figure 3B). This protein was expressed in a similar 206 stochastic pattern observed for active caspase-3 and its expression was also reduced by p38α/β 207 inhibition ( Figure 4B and C). Our results demonstrate that DUX4 inhibition in FSHD myotubes 208 results in a significant reduction of apoptosis.       Myotubes were grown in 96-well plates using conditions described above and were lysed using 452 25 μL of 1X MSD lysis buffer with protease and phosphatase inhibitors. The lysates were 453 incubated at room temp for 10 minutes with shaking at 1200 rpm using Titramax 1000. Lysates 454 were stored at -80 o C until all timepoints were collected. Lysates were then thawed on ice and 2 455 μL were used to perform a BCA protein assay (ThermoFisher, # 23225). 10 μL of lysate were 456 diluted 1:1 in 1X MSD lysis buffer and added to the 96-well Mesoscale assay plate. Manufacturer 457 instructions were followed, and data was obtained using a MesoScale Discovery SECTOR S 600 458 instrument. 459

Caspase-3 478
Myotubes were grown and treated as described above. At day 5 after differentiation was induced, 479 cells were fixed using 4% paraformaldehyde in PBS during 10 min at room temperature. Fixative 480 was washed, and cells were permeabilized using 0.5% Triton X-100 during 10 min at room 481 temperature. After washing, fixing and permeabilizing, the cells were blocked using 5% donkey 482 serum in PBS/0.05% Tween 20 during 1 h at room temperature. Primary antibodies against MHC 483 (MF20, R&D systems, #MAB4470), SLC34A2 (Cell signaling, #66445) and active Caspase-3 (Cell 484 signaling, #9661) were diluted 1:500 in PBS containing 0.1% Triton X-100 and 5% donkey serum 485 and incubated with cells for 1 h at room temperature. After 4 washes, secondary antibodies were 486 added (ThermoFisher, #A32723 and # R37117) in a 1:2000 dilution and incubated during 1 h at 487 room temperature. During the last 5 min of incubation a 1:2000 dilution of DAPI was added before 488 proceeding with final washes and imaging. Images were collected using the CellInsight CX7 489 (ThermoFisher). Images were quantified using HCS Studio Software. Differentiation was 490 quantified by counting the percentage of nuclei in cells expressing MHC from the total of the well. 491

SLC34A2 and active Caspase-3 signal was quantified by colocalization of cytoplasmic cleaved 492
Caspase-3 within MHC expressing cells. 493

Knockdown of MAPK12 and MAPK14 in FSHD myotubes 494
Exponentially dividing immortalized C6 FSHD myoblasts were harvested and counted. 50000 495 myoblasts were electroporated using a 10 μL tip in a Neon electroporation system 496 (ThermoFisher). Conditions used were determined to preserve viability and achieved maximal 497 electroporation (Pulse V=1100V, pulse width=40 and pulse #=1). After electroporation, cells were 498 plated in growth media and media was changed for differentiation 24h after. 3 days after 499 differentiation, cells were harvested and analyzed for KD and effects in MBD3L2 using the RT-500 qPCR assay described before. siRNAs used were obtained from ThermoFisher (4390843, 501 4390846, s3585, s3586, s12467, s12468). 502

Gene expression analysis by RNA-seq 503
RNA from myotubes grown in 6-well plates in conditions described above was isolated using the 504 RNeasy Micro Kit from Qiagen (#74004). Quality of RNA was assessed by using a Bioanalyzer 505 2100 and samples were submitted for library preparation and deep sequencing to the Molecular 506 biology core facility at the Dana Farber Cancer Institute. After sequencing, raw reads of fastq files 507 from all samples were mapped to hg38 genome assemblies using ArrayStudio aligner. Raw read 508 count and FPKM were calculated for all the genes, and DESeq2 was applied to calculate 509