EP.321 Revolutionizing drug discovery in genetically ...
1
Characterization of Novel Targets in FSHD Our vision at Fulcrum Therapeutics is to transform the future of patients living with genetically-defined diseases. Our approach to drug discovery avoids the pitfalls that have plagued our industry and harnesses recent technological advances that allow us to forge a new path for patients. Using the latest advances in single cell methods, we can look deeper into diseased tissue to generate powerful insights into the drivers of disease and identify new ways to re-balance the cellular composition and dysregulation of pathological microenvironments. Our FulcrumSeek platform brings together the deep understanding of tissue biology elucidated from the hypothesis-generating combination of snRNA-seq and spatial transcript/proteomics studies of human samples, with the latest technologies in cellular modeling and comprehensive phenotypic screening using high-throughput RNA-seq and high content imaging. We are applying the power of machine learning together with the creative and inventive spark of human intelligence to unravel the richness of this data and revolutionize drug discovery in muscular dystrophies. Using this approach, we have characterized biopsies from dystrophic muscle to understand the cellular composition, interactions, and processes that drive muscle degeneration and loss of function. Now, we are creating a database of cellular profiles generated in response to our highly curated and annotated chemical library and functional genomics tools to identify novel targets that can modulate these drivers of disease. Here, we demonstrate that by applying this integrated target discovery approach in FSHD patient biopsies as well as patient-derived myotubes in vitro, we can rapidly rediscover previously known targets in FSHD, as well as identifying novel targets. This platform has the potential to fundamentally change the way drugs are discovered and developed for patients with devastating muscle diseases. Abstract Approach to Target Discovery at Fulcrum Therapeutics Facioscapulohumeral Muscular Dystrophy (FSHD) L. Alejandro Rojas, Ayla Ergun, Caroline Sartain, Anthony Accorsi, Richard Lieberman, Anugraha Raman, Joe Maglio, Erin Valentine, Mary Wertz, Elizabeth Townsend, Damon Polioudakis, Ivan Efremov, Lorin A. Thompson, Serena J. Silver, Christopher M. Moxham Fulcrum Therapeutics. 26 Landsdowne Street, Cambridge, Massachusetts, USA. Revolutionizing drug discovery in genetically-defined muscle disease using single-cell and high-dimensional datasets Treatments that Modulate Individual Disease Genes Expression INCREASE DECREASE Treatments that Revert Gene Signatures of Disease Disease Signature With Treatment Treatments that Modulate Cell Fate in Diseases Disease Tissue With Treatment FulcrumSeek Platform identifies targets that have the potential to rebalance monogenic, polygenic, and heterogenous diseases Our core focus is on patients with diseases of high unmet need We use the most relevant cellular models to study a given disease We systematically employ target & modality agnostic screening approaches to enable rapid identification of high-quality targets Fulcrum’s patient-centric approach systematically & rapidly pinpoints multiple novel intervention points FulcrumSeek TM : Accelerating Discovery of Disease-Modifying Therapies FulcrumSeek TM Product Engine enables intelligent drug discovery in disease-relevant models through high-dimensional data and machine learning Computational Engine Machine Learning on high dimensional transcriptomic and imaging data enables insights into target and disease biology Accelerated Drug Discovery Programs within each Tissue Discovery Engine Insights from proprietary clinical samples / datasets Disease relevant cell models interrogated with small molecules, RNAi and CRISPR Targets with specificity, selectivity and tolerability Tissue-relevant translatable biomarkers Foresight on potential toxicity or off-target activity Highly de-risked target and development candidates Disease Healthy Ineffective Treatment Desired Phenotype Isogenic Control Single Cell Genomics High-throughput transcriptomic profiling High-content imaging for morphological profiling FulcrumSeek HT-3'RNA-seq Significant advantages over published state-of-the- art methods Optimized at each step based on empirical data Universal workflow suitable for all tissue-relevant cell models Cost-effective workflow optimized for automation and decreased processing time Comparable screening metrics to conventional assays (qPCR, ICC, MSD) Multiplexed readout endpoint for comprehensive phenotypic screening ~8-10K transcripts per well in 384w plate format 384w plate level visualization of QC metrics used before data analysis Transcriptional changes induced by compound treatments cluster by target and pathways Aligned reads per well Duplication rate per well Number of genes detected Segmentation and extraction of cellular features for incorporation of visual, feature, and expression data into computational pipeline to characterize phenotypic changes induced by perturbations Nucleus Cell-specific Mitochondria ER F-Actin Nucleus Cell-specific Mitochondria Endoplasmic Reticulum F-Actin Cardiomyocytes Myotubes Neurons Microglia Astrocytes This approach allows for the identification and characterization of phenotypic changes in myotubes caused by small molecule, functional genomic perturbations, and disease states. DMSO Compound A Compound B Compound C Compound D Wildtype myoblasts were treated with indicated compounds and differentiated for 7 days in vitro before fixation and staining Developing Novel Technologies for Phenotypic Screening FSHD is a debilitating, relentlessly progressive neuromuscular disease with heterogeneous muscle involvement and variable onset, severity, and progression. FSHD is characterized by progressive skeletal muscle weakness affecting the face, shoulders, arms, trunk, weakness of the lower extremities and pelvic girdle. Symptoms typically appear in the second decade but can begin at any age. FSHD is caused by the aberrant expression of DUX4 in skeletal muscle. This transcription factor is usually only expressed in early embryonic development Stochastic DUX4 expression results in heterogenous presentation between patients and within muscles in the same patient. An ideal target that downregulates DUX4 will decouple its expression and the myogenesis/regeneration process Fulcrum has previously identified the p38α/β inhibitor losmapimod as a potential therapeutic for FSHD DUX4 protein DUX4 targets DUX4 ON P38α/β • Regulates expression of DUX4 gene DUX4 protein • Activates expression of DUX4 targets genes during embryogenesis FSHD • Loss of repression in D4Z4 repeats allows for aberrant DUX4 expression in muscle DUX4 expression and activity • In FSHD, aberrant expression in muscle causes muscle cell death and immune and fat infiltration Progressive muscle loss and fatty replacement Progressive worsening of function Using our high-dimensional approach we can rapidly identify and prioritize the highest quality targets that result in desired phenotypic changes and have no undesired effects in other biological processes DUX4 is lowly expressed and is not detectable by HT-3’ RNAseq, but a set of DUX4 program genes can be used as a signature of DUX4 activity. Inhibition of p38α/β results in the inhibition of DUX4 expression and minimal effects in myogenesis. Inhibition of novel targets that reduce DUX4 without impacting myogenesis can be identified and quickly prioritized for further characterization and validation Needle Biopsy Collection 16 FSHD STIR+ biopsies 2 Healthy biopsies taken Nuclei prep/isolation from flash frozen tissue and snRNA-seq 126k nuclei from disease biopsies ~23 k healthy biopsies Downstream Processing of snRNA-seq data Characterization of cell types Canonical marker genes Published gene signatures Gene enrichment Characterization of cell type proportions Differences between Healthy/FSHD Generating a cellular atlas of skeletal muscle tissue in FSHD to identify the most relevant cell types for disease modelling in vitro Adipocytes FAP Macrophage SCs Pericytes Smooth Muscle B Cell Endothelial Myonuclei T Cell Myonuclei Fibroblasts Myonuclei ~ 89 k nuclei DUX4 Target expressing cells ~ 30 cells ~6.3k myonuclei subtype Cells associated with regeneration/myogenesis expressing markers like MYH3 1 in 3000 myonuclei have detectable DUX4 activity using snRNA-seq profiling Identifying DUX4-expressing cells in FSHD skeletal muscle Losmapimod reduced p38 activity, DUX4 expression, DUX4 activity, and cell death in patient-derived FSHD myotubes HSP27 is a substrate of p38 MAP kinase pathway MBD3L2 is a DUX4-target gene Caspase-3 is an indicator of cell death Preclinical study demonstrated a reduction of DUX4 activity across multiple patient-derived FSHD1/2 myotubes 0.001 0.01 0.1 1 0 25 50 75 100 125 % of DMSO control Losmapimod (μM) n=8 n=3 Losmapimod Probe library ~2000 targets 6000 compounds CRISPR/siRNA Library ~5000 selected targets Target space Small molecule probe library ~6000 hand-selected small molecules from literature, patents and commercial sources Constantly being updated with newly synthesized compounds of interest ~2000 targets with diverse mechanisms, including kinase, chromatin modifiers, nuclear hormone receptors, GPCRs, ion channels and other enzymes. CRISPR gRNA and siRNA libraries ~5000 prioritized targets including mechanisms of interest such as signaling pathways, chromatin modifiers, transcription factors. Opportunity for identification of targets with no chemical matter available Scalable miniaturized delivery protocols optimized for challenging cell models in arrayed screening formats Chemical Biology Approach for the Rapid Identification of Tractable Targets 0.001 0.01 0.1 1 0 25 50 75 100 125 150 % of DMSO control DUX4 target Compound X (µ M) Target engagement Active Caspase-3 Differentiation index Novel Target X inhibition results in potent reduction of DUX4, its activity, and its downstream consequences DMSO Compound X (0.1uM) p38 inhibitor (1uM) 0.0 0.5 1.0 1.5 Fold Change to DMSO MBD3L2 DUX4fl Immunofluorescence analysis of indicated endpoints after 7 days of treatment and differentiation of FSHD myotubes RT-qPCR analysis of indicated endpoints after 7 days of treatment and differentiation of FSHD myotubes Increasing concentration of Compound X FSHD Myotubes Nucleus MHC SLC34A2 (DUX4 target) Enabling Unprecedented Drug Discovery at Scale in Disease-Relevant Settings Embodies disease context and focuses on root cause of disease Enabled to pursue numerous indication areas: musculoskeletal, hematology, cardiovascular, CNS, pulmonary, and many others Represents the nexus of state-of-the-art advances in high content technologies, machine learning, cellular modeling, and pharmacogenomics Comprehensive approach to rebalance diverse disease states for disease-modifying therapies Targets, pathways, cell types, therapeutic areas Our Discovery Platform EP.321
Transcript of EP.321 Revolutionizing drug discovery in genetically ...
Characterization of Novel Targets in FSHDOur vision at Fulcrum Therapeutics is to transform the future of patients living with genetically-defined diseases. Ourapproach to drug discovery avoids the pitfalls that have plagued our industry and harnesses recent technologicaladvances that allow us to forge a new path for patients. Using the latest advances in single cell methods, we canlook deeper into diseased tissue to generate powerful insights into the drivers of disease and identify new ways tore-balance the cellular composition and dysregulation of pathological microenvironments. Our FulcrumSeek platformbrings together the deep understanding of tissue biology elucidated from the hypothesis-generating combination ofsnRNA-seq and spatial transcript/proteomics studies of human samples, with the latest technologies in cellularmodeling and comprehensive phenotypic screening using high-throughput RNA-seq and high content imaging. Weare applying the power of machine learning together with the creative and inventive spark of human intelligence tounravel the richness of this data and revolutionize drug discovery in muscular dystrophies. Using this approach, wehave characterized biopsies from dystrophic muscle to understand the cellular composition, interactions, andprocesses that drive muscle degeneration and loss of function. Now, we are creating a database of cellular profilesgenerated in response to our highly curated and annotated chemical library and functional genomics tools to identifynovel targets that can modulate these drivers of disease. Here, we demonstrate that by applying this integratedtarget discovery approach in FSHD patient biopsies as well as patient-derived myotubes in vitro, we can rapidlyrediscover previously known targets in FSHD, as well as identifying novel targets. This platform has the potential tofundamentally change the way drugs are discovered and developed for patients with devastating muscle diseases.
Abstract
Approach to Target Discovery at Fulcrum Therapeutics
Facioscapulohumeral Muscular Dystrophy (FSHD)
L. Alejandro Rojas, Ayla Ergun, Caroline Sartain, Anthony Accorsi, Richard Lieberman, Anugraha Raman, Joe Maglio, Erin Valentine, Mary Wertz, Elizabeth Townsend, Damon Polioudakis, Ivan Efremov, Lorin A. Thompson, Serena J. Silver, Christopher M. Moxham
Fulcrum Therapeutics. 26 Landsdowne Street, Cambridge, Massachusetts, USA.
Revolutionizing drug discovery in genetically-defined muscle disease using single-cell and high-dimensional datasets
Treatments that Modulate Individual Disease Genes
Expr
essi
on
INCREASE DECREASE
Treatments that Revert Gene Signatures of Disease
DiseaseSignature
WithTreatment
Treatments that Modulate Cell Fate in Diseases
DiseaseTissue
WithTreatment
FulcrumSeek Platform identifies targets that have the potential to rebalance monogenic, polygenic,
and heterogenous diseases
Our core focus is on patients with diseases of high unmet need
We use the most relevant cellular models to study a given disease
We systematically employ target & modality agnostic screening approaches to enable rapid identification of high-quality targets
Fulcrum’s patient-centric approach systematically & rapidly pinpoints multiple novel intervention points
FulcrumSeekTM: Accelerating Discovery of Disease-Modifying Therapies
FulcrumSeekTM Product Engine enables intelligent drug discovery in disease-relevant models through high-dimensional data and machine learning
Computational Engine
Machine Learning on high dimensional transcriptomic and
imaging data enables insights into target and disease biology
Accelerated Drug Discovery Programswithin each Tissue
Discovery Engine
Insights from proprietary clinical samples / datasets
Disease relevant cell models interrogated with small molecules, RNAi
and CRISPR
Targets with specificity, selectivity and tolerability
Tissue-relevant translatable biomarkers
Foresight on potential toxicity or off-target activity
Highly de-risked target and
development candidates
Disease
HealthyIneffective Treatment
Desired Phenotype
Isogenic Control
Single Cell Genomics
High-throughput transcriptomic profiling
High-content imaging for morphological profiling
FulcrumSeek HT-3'RNA-seqSignificant advantages over published state-of-the-art methods Optimized at each step based on empirical data Universal workflow suitable for all tissue-relevant
cell models Cost-effective workflow optimized for automation
and decreased processing time Comparable screening metrics to conventional
assays (qPCR, ICC, MSD)
Multiplexed readout endpoint for
comprehensive phenotypic screening
~8-10K transcripts per well in 384w plate format
384w plate level visualization of QC metrics used before data analysis
Transcriptional changes induced by compound treatments cluster by target and pathways
Aligned reads per well Duplication rate per well
Number of genes detected
Segmentation and extraction of cellular features for incorporation of visual, feature, and expression data into computational pipeline to characterize phenotypic changes induced by perturbations
NucleusCell-specificMitochondriaERF-Actin
Nucleus Cell-specific Mitochondria Endoplasmic Reticulum F-Actin
Cardiomyocytes Myotubes Neurons Microglia Astrocytes
This approach allows for the identification and characterization of phenotypic changes in myotubes caused by small molecule, functional genomic perturbations, and disease states.
DMSO Compound A Compound B Compound C Compound D
Wildtype myoblasts were treated with indicated compounds and differentiated for 7 days in vitro before fixation and staining
Developing Novel Technologies for Phenotypic Screening
FSHD is a debilitating, relentlessly progressive neuromuscular disease with heterogeneous muscle involvement and variable onset, severity, and progression.
FSHD is characterized by progressive skeletal muscle weakness affecting the face, shoulders, arms, trunk, weakness of the lower extremities and pelvic girdle. Symptoms typically appear in the second decade but can begin at any age.
FSHD is caused by the aberrant expression of DUX4 in skeletal muscle. This transcription factor is usually only expressed in early embryonic development
Stochastic DUX4 expression results in heterogenous presentation between patients and within muscles in the same patient.
An ideal target that downregulates DUX4 will decouple its expression and the myogenesis/regeneration process
Fulcrum has previously identified the p38α/β inhibitor losmapimod as a potential therapeutic for FSHD
DUX4protein DUX4 targets
DUX4
ON
P38α/β• Regulates
expression of DUX4 gene
DUX4 protein• Activates expression of DUX4 targets genes during
embryogenesis
FSHD• Loss of repression in D4Z4 repeats allows for
aberrant DUX4 expression in muscle
DUX4 expression and activity• In FSHD, aberrant expression in muscle causes muscle cell
death and immune and fat infiltration
Progressive muscle loss and fatty replacement
Progressive worsening of function
Using our high-dimensional approach we can rapidly identify and prioritize the highest quality targets that result in desired phenotypic changes and have no
undesired effects in other biological processes
DUX4 is lowly expressed and is not detectable by HT-3’ RNAseq, but a set of DUX4 program genes can be used as a signature of DUX4 activity.
Inhibition of p38α/β results in the inhibition of DUX4 expression and minimal effects in myogenesis.
Inhibition of novel targets that reduce DUX4 without impacting myogenesis can be identified and quickly prioritized for further characterization and validation
Needle Biopsy Collection 16 FSHD STIR+
biopsies 2 Healthy biopsies
taken
Nuclei prep/isolation from flash frozen tissue and snRNA-seq 126k nuclei from disease
biopsies ~23 k healthy biopsies
Downstream Processing of snRNA-seq dataCharacterization of cell typesCanonical marker genesPublished gene signaturesGene enrichmentCharacterization of cell type
proportions Differences between
Healthy/FSHD
Generating a cellular atlas of skeletal muscle tissue in FSHD to identify the most relevant cell types for disease modelling in vitro
Adipocytes
FAP
Macrophage
SCs
Pericytes
Smooth Muscle
B Cell
Endothelial
MyonucleiT Cell
Myonuclei
Fibroblasts
Myonuclei ~ 89 k nuclei DUX4 Target expressing cells~ 30 cells
~6.3k myonuclei subtype
Cells associated with regeneration/myogenesis expressing markers like
MYH3
1 in 3000 myonuclei have detectable DUX4 activity using
snRNA-seq profiling
Identifying DUX4-expressing cells in FSHD skeletal muscle
Losmapimod reduced p38 activity, DUX4 expression, DUX4 activity, and cell death in patient-derived FSHD myotubes
HSP27 is a substrate of p38 MAP kinase pathway MBD3L2 is a DUX4-target gene Caspase-3 is an indicator of cell death
Preclinical study demonstrated a reduction of DUX4 activity across multiple patient-derived FSHD1/2 myotubes
0.001 0.01 0.1 10
25
50
75
100
125
% o
f DM
SO c
ontro
l
Losmapimod (μM)
n=8n=3
Losmapimod
Probe library~2000 targets6000 compounds
CRISPR/siRNA Library~5000 selected targets
Target space
Small molecule probe library ~6000 hand-selected small molecules from literature, patents and
commercial sources Constantly being updated with newly synthesized compounds of interest ~2000 targets with diverse mechanisms, including kinase, chromatin
modifiers, nuclear hormone receptors, GPCRs, ion channels and other enzymes.
CRISPR gRNA and siRNA libraries ~5000 prioritized targets including mechanisms of interest such as
signaling pathways, chromatin modifiers, transcription factors. Opportunity for identification of targets with no chemical matter available Scalable miniaturized delivery protocols optimized for challenging cell
models in arrayed screening formats
Chemical Biology Approach for the Rapid Identification of Tractable Targets
0.001 0.01 0.1 10
25
50
75
100
125
150
% o
f DM
SO c
ontro
l
DUX4 target
Compound X (µM)
Target engagementActive Caspase-3Differentiation index
Novel Target X inhibition results in potent reduction of DUX4, its activity, and its downstream consequences
DMSO
Compound X (0.1u
M)
p38 in
hibitor (1
uM)0.0
0.5
1.0
1.5
Fold
Cha
nge
to D
MSO MBD3L2
DUX4fl
Immunofluorescence analysis of indicated endpoints after 7 days of treatment and
differentiation of FSHD myotubes
RT-qPCR analysis of indicated endpoints after 7 days of treatment and
differentiation of FSHD myotubes
Increasing concentration of Compound X
FSH
D M
yotu
bes
Nucleus MHC SLC34A2 (DUX4 target)
Enabling Unprecedented Drug Discovery at Scale in Disease-Relevant Settings
Embodies disease context and focuses on root cause of diseaseEnabled to pursue numerous indication areas: musculoskeletal, hematology,
cardiovascular, CNS, pulmonary, and many others
Represents the nexus of state-of-the-art advances in high contenttechnologies, machine learning, cellular modeling, and pharmacogenomics
Comprehensive approach to rebalance diverse disease states fordisease-modifying therapies
Targets, pathways, cell types, therapeutic areas
Our Discovery Platform
EP.321
