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Fulcrum WMS DUX4 FSHD Poster Targeting DUX4 Expression, the Root Cause of FSHD: Identification of a Drug Target and Development Candidate Owen B. Wallace, Anthony Accorsi, Richard Barnes, Angela Cacace, Diego Cadavid, Aaron Chang, David Eyerman, Robert Gould, Steven Kazmirski, Joseph Maglio, Michelle Mellion, Peter Rahl, Alan Robertson, Alejandro Rojas, Lucienne Ronco, Ning Shen, Lorin A. Thompson and Erin Valentine Fulcrum Therapeutics. 26 Landsdowne Street, Cambridge, Massachusetts, USA. Abstract 4. Losmapimod reduces DUX4 activation and its downstream consequences • FSHD is caused by the loss of repression at the D4Z4 locus leading to aberrant DUX4 expression A. 150 in skeletal muscle, activation of its early embryo transcriptional program and muscle fiber death. 125 100 • While some progress toward understanding the signals driving DUX4 expression has been made, 75 the factors and pathways involved in the transcriptional activation of this gene remain largely 50 unknown. 25 (% of nuclei in MHC mask) MHC in nuclei of (% % of DMSO% of control 0 • Using optimized myotube culture conditions, we identified p38 MAPK as a key regulator of DUX4 0.001 0.01 0.1 1 expression. [Losmapimod] µM • We observed that treatment with the p38α/β inhibitor losmapimod results in reduction of DUX4 B. C. expression, activity and cell death in FSHD patient-derived myotubes with minimal impact on WT FSHD myogenesis. 125 MBD3L2 mRNA 100 • RNA-seq studies revealed that only a small number of genes were differentially expressed after Active Caspase-3 treatment with losmapimod, ~90% of these are targets of DUX4. 75 50 • Fulcrum Therapeutics has selected losmapimod, a specific p38α/β inhibitor, for clinical 25 development in FSHD. DMSO% of control 0 0.001 0.01 0.1 1 Cleaved Caspase-3 MHC DAPI [Losmapimod] µM 1. Facioscapulohumeral Muscular Dystrophy (FSHD) A. B. (A) Losmapimod treatment does not affect in vitro differentiation of myotubes. (B) RNA-seq analysis of myotubes indicates that losmapimod selectively inhibits DUX4 and its downstream program expression in a concentration dependent manner with minimal impact across the transcriptome of FHSD myotubes. <100 differentially expressed genes (abs(FC)>4; FDR<0.001) (C) Losmapimod reduces cleaved caspase-3 signal detected in FSHD myotubes indicating reduction of cell death. 5. DUX4 activity and apoptosis in primary myotubes A. B. FSHD1 FSHD2 1800 (A) Schematic of the loss in gene repression caused by contraction of D4Z4 repeats that leads to DUX4 1600 1400 expression in the muscle of FSHD1 patients. 1200 Schematic describing the downstream consequences of DUX4 expression in skeletal muscle. 1000 (B) 800 600 (% of POLR2A) 400 200 MBD3L2 mRNA MBD3L2 2. Fulcrum’s approach to target identification and validation 0 DAPI MHC Cleaved Caspase-3 WT Control CTID-003CTID-005 (3) (2) CTID-009CTID-010 (2) CTID-012(3) (7) CTID-002 (5.5)CTID-006CTID-007 (4.5) (4.5) CTID-001CTID-013 CTID-004(WT) (WT)FSHD (WT)FSHD C6 (6.5) C12 (3) CTID-008 (SMCHD1)CTID-011 CTID-014(SMCHD1) (SMCHD1) Cell Biology Target ID Target Physiology (A) MBD3L2 expression across a variety of primary FSHD patient- and non-FSHD (WT) derived Small molecule and myotubes. (B) Detection of cleaved caspase-3 in primary FSHD1 and FSHD2 patient-derived CRISPR/Cas9 Gene expre ssion Phenotype models screening myotubes. Cell-based screening drives Fulcrum’s target identification engine 6. Studying p38 inhibition across FSHD genotypes “Clinical trial in a dish” • 11 primary FSHD cell lines: 8 FSHD1 and 3 FSHD2. • 3 non-FSHD cells as negative control and one immortalized FSHD cell as positive control. • Primary Efficacy Analysis: Wilcoxon matched-pairs signed rank test was perform between different 3. Identification of a drug target that inhibits DUX4 expression treatment groups and vehicle group samples to determine significant differences. DUX4 mRNA MBD3L2 mRNA A. B. A. B. 0.04 35 Day 5 30 0.03 25 Day -2 Day 0 Day 2 20 Wilcoxon matched-pairs signed rank test 0.02 15 to HMBS 0.01 10 Seeding Fusion Relative expression 5 Myotubes 0.00 0 Differentiation media and compound treatment Bright field ACTN2 + DAPI WT day 5 WT Day 5 FSHDFSHD Day FSHD0 Day FSHD1 Day 3 Day 5 FSHDFSHD Day FSHD0 Day FSHD1 Day 3 Day 5 C. 1.50 D. E. FSHD myotubes DMSO C. 1.25 All FSHD FSHD mt + DUX4 ASO 1.00 FSHD1 FSHD myoblasts signal WT myotubes 2 0.75 L FSHD2 3 0.50 Robust Z’: 0.512 0.25 MBD S/N: 5.1 Relative to control to Relative 0.00 Hits DUX4 WT ASO [FTX-2865] [FTX-2865] F. G. H. 125 120 MAPK14 mRNA MBD3L2 mRNA ✱ DUX4 protein 150 150 100 100 ✱✱✱ (A) Table describing the genotypes of cells used in this study. (B) MBD3L2 expression was MBD3L2 mRNA 125 125 80 75 100 100 reduced across all FSHD1 and FSHD2 patient-derived myotubes after losmapimod treatment. 60 75 75 50 (C) Inhibition of p38α/β MAPK pathway using a structurally distinct inhibitor (FTX-2865) results in 40 50 50 25 25 25 reduction of DUX4 expression and inhibition of cell death in vitro across FSHD1 and FSHD2 p38 pathway activity % of DMSO control ofDMSO % 20 0 0 % of NT CTRL Mean CTRL NT % of 0 DMSO% of control 0 genotypes. 0.001 0.01 0.1 1 0.001 0.01 0.1 1 [Losmapimod] µM [Losmapimod] µM NT CTRLNT 1CTRL 2 NT CTRLNT 1 CTRL 2 siMAPK14siMAPK14 85 86 siMAPK14siMAPK14 85 86 6. Conclusions (A) Cell-based assay schematic. (B) Expression of DUX4 and DUX4 target gene MBD3L2 during • Using an in vitro model of FSHD, we identify novel regulators of aberrant DUX4 expression. FSHD myotube differentiation in vitro. (C) DUX4 target gene selection for HTS. (D) 96-well format • Losmapimod is a selective p38α/β inhibitor that reduces DUX4 expression in FSHD myotubes. assay allows for identification of targets modulating the expression of DUX4. (E) Hits identified • Further in vitro characterization demonstrates that the DUX4-driven gene expression and cell showed high correlation in between biological replicates. (F) Losmapimod reduces expression of death are inhibited in FSHD myotubes exposed to losmapimod. DUX4 in a concentration dependent manner in FSHD myotubes. (G) Losmapimod reduces p38 • Losmapimod reduced expression of DUX4 in all FSHD1 and FSHD2 cell lines tested. α/β activity in FSHD myotubes. (H) P38α knockdown reduces activity of DUX4 in FSHD myotubes. • Reduction of DUX4 expression resulted in inhibition of cell death across all genotypes tested..
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