progress and future directions in integrated …...from moa studies validated pathway assay (stem)...
TRANSCRIPT
Mary McBride
Agilent Technologies
1 September 30, 2014
Progress and Future Directions in
Integrated Systems Toxicology
2
Toxicity testing tools of the late 20th century
High Dose Testing in Animals with Extrapolation to Human-Relevant Doses
• Time-consuming and expensive ($3B/year)
• Requires exorbitant use of animals
• Large number of endpoints
• Not amenable to high throughput
• Questionable relevance to humans
• Conservative extrapolation tools
September 30, 2014
Patchwork approach to testing dates back to the 1930’s
3
Toxicity testing in the 21st century NRC report provided a vision and a strategy for shift to in vitro testing
NRC Report, 2007
Report Called for a Transformative Change to Toxicity Testing, with 4 Major Components:
Chemical Characterization: Physical and chemical properties, use, exposure routes, metabolites
Toxicity Pathways: Employ high-throughput cell-based assays (of human origin) with integrated ‘omics measurements to evaluate perturbations to relevant toxicity pathways (systems biology approach)
Targeted Testing: Conduct limited and directed testing using whole animals only until in vitro methods reliably predict outcomes
Dose-Response: Couple assay data with computational systems biology to address dose-response and in vitro-to-in vivo extrapolations
September 30, 2014
4
TT21C in practice: Systems toxicology approach Can provide deep mechanistic understanding of toxicological effects, enable prediction
September 30, 2014
Sturla , et.al. Systems Toxicology: from basic
research to risk assessment. Chem. Res.
Toxicol., 27:314-329. (PMID: 24446777).
Systems Toxicology is:
the integration of the classic toxicology paradigm with the quantitative analysis of many molecular and functional changes occurring across multiple levels of biological organization.
5
xxx
Systems biology to map and model pathways Using known toxicity pathways to rapidly go from assays to risk assessments
September 30, 2014
Computational Systems Biology Pathway Model
In-vitro to in-vivo extrapolation
Compare to in-vivo
outcomes from MOA
studies
Validated Pathway Assay
(Stem) cell biology, high-throughput pathway assays
Multi-omics pathway analysis
Time course, dose response
Other data streams: Ca transients,
cell imaging, etc
Computational cell biology, control
theory, systems dynamics
6
Cell-based assays for predictive toxicology In-vitro platforms for drug and chemical safety, toxicity testing and disease modeling
September 30, 2014
Cardiomyocytes Chondrocytes Hepatocytes
• Proliferate extensively • Differentiate into any cell type • Recapitulate embryonic processes, providing
insights into development “windows” • Enable access to population-level phenotypes • Ethical/legal considerations (adult, induced) • Effects unknown; Long term studies needed
Primary Human Cells
• Limited availability • Variable quality • Phenotypic instability • Donor variability • Limited characterization
Human Stem Cells
Transformed Cell Lines
• May not recapitulate cell/tissue biology
• May lack key functional characteristics
• Inadequately represents human diversity
Animal Models
• Represent human biology?
• Resource intensive • Animal welfare issues
7
3D culture simulates in-vivo cell environment Innovations in 3D culture are enabling more biologically-relevant results
September 30, 2014
2D 3D
• Rigid inert substrates • Cells partially
interact •Not representative of
in vivo environment
• Porous, flexible (ECM gels) • Extensive cell-cell
communication and signaling • Better representation of in vivo environment with
micro-engineered controls
2D Cell Culture 3D Cell Culture
DARPA/FDA/NIH “Human-on-a-Chip”
Integrating “organ on a chip” microdevices to produce physiologically and pathologically accurate models of human organ systems
8
Cell-based imaging Enables visualization of molecular, biochemical, and cellular processes in living cells
September 30, 2014
• GFP enabled live cell labeling, multiple colors
• Non-destructive technique, can image in 3D culture
• View/quantify cellular dynamics in real time
• Advances in auto-focusing, sample positioning, SW
• Functional and morphological detail on individual cells, not “averages”
Slide from Chad Deienroth, Hamner Institutes
9
Next-gen tailored in-vitro fit-for-purpose assays Need in-vitro assays to map pathway circuitry, understand perturbations and MOAs
September 30, 2014
Various high throughput tests
Pr + L Pi - L
Reporter assay
Evaluate Results
Determine consistency of
observed MOAs and expected
targets
e.g., most likely reproductive
toxicity through E2 activation
Move on to
specific in vitro pathway assays for E2-pathway
Safety
Assessment E2-Pathway
AhR-Pathway
DNA-damage Pathway
Oxidative Stress Pathway
Mitochondrial Damage Pathways
“Validated” Toxicity Pathway Assays
CSBP Models in Vitro PK
EC10
Slide courtesy Mel Andersen, Hamner Institute , 2013
10
Automated high-throughput screening Tox21C has demonstrated rapid screening of in-vitro assays
September 30, 2014
• Each compound tested against battery of >700 biochemical and cell-based assays
• 15 concentration points, triplicates and controls
• Phase II assay panel to include more pathway-based assays (e.g., nuclear receptor, oxidative stress)
• Miniaturized assay volumes 2-6 uL in 1536-well plate
• Informatics pipeline for data processing, curve fitting & classification, extraction of SAR
Other assay initiatives:
• CRISPR technology for genome editing
• HTS R-qPCR (liquid robotics, sample prep reeagents and protocols
• Stem cells to correlate in vitro phenotype to clinical phenotype
11
Multi-omics pathway analysis of dose-response Illuminating biological understanding through a systems biology approach
September 30, 2014
Production
Regulation
Genomics Proteomics Transcriptomics Metabolomics
Genes mRNA Protein Metabolite
Prevailing paradigm for biological information flow does not fully describe the system
12
Multi-omics analysis in GeneSpring Pathway-centric approach to multi-omics research powered by GeneSpring Analytics
September 30, 2014
13
Visualize individual samples with their metadata information
September 30, 2014
GeneSpring Metadata Framework (Ver. 15.xx)
Can we unambiguously link adverse outcome phenotype to underlying MOA?
14
A private sector partnership for toxicity testing Partnership is accelerating implementation of in vitro tox testing
–One representative from each partner –Development and oversight of research strategy and partner
interactions –Administrative budgeting, organization, etc.
Governance Board
EPA- Regulatory/policy, risk assessment, computational models and database hosting. NIH, FDA may be better positioned to join in 2014.
Provide instrumentation and expertise for genomic, metabolomic, proteomic, and transcriptomic studies, bioinformatics, data analysis and visualization tools
Technology
Direct and coordinate laboratory R&D. Develop assays and protocols. Analyze data. Work with public and private
partner in developing software and bioinformatics tools.
Academic Thought Leaders
Industry
Leverage $3M investments already made for Hamner estrogen-signaling case study approach; technology sector investments to extend/accelerate R&D
September 30, 2014
15
Case studies using prototype pathways Using known toxicity pathways to rapidly go from assays to risk assessments
September 30, 2014
1. Select well-studied prototype compounds targeting specific pathways Estrogen receptor mediated proliferation p53-DNA-damage and mutation PPAR-a and lipid metabolism Nrf2-Keap1 oxidative stress AhR liver induction and altered cell growth mitochondrial stress and toxicity
2. Design cell-based toxicity pathway assays to understand key portions of
the network that control dose-response behaviors
3. Refine new quantitative risk assessment tools, i.e., computational pathway models and in vitro to in vivo extrapolation
4. Integrate results into proposed health safety/risk assessments.
16
computational systems biology pathway (CSBP) models for
predicting acceptable exposures
A systems toxicology approach to PPAR-a pathway
September 30, 2014
• Human and rat primary hepatocytes treated with PPARα agonist, GW7647
at 5 doses and 5 time points
• Microarrays for gene expression
• Protein-DNA Arrays for DNA binding
• Evaluate transcription factor network
• Examine functional relationships of regulated genes
• Measure metabolomic signatures in ‘secretome’
Developing a testing strategy for nuclear-receptor mediated proliferation
17
Activation of liver nuclear receptors Expanding the map of nuclear reception activation for PPAR-a
Slide Courtesy Mel Andersen, Hamner Institute for Chemical Safety Sciences
September 30, 2014
Summary of findings to date:
• Transcription results after GW7647: Rat (971 genes); Human (192 genes);
lipid metabolism gene responses (54 genes) conserved between two
species; large number down-regulated genes in rat.
• ChIP-seq results show: Direct binding of PPARa accounts for only half of
genes. Alternate binding mode(s) found for rat, but not for humans:
PPARa associates with promoters of down-regulated genes; binding
regions differ for up vs. down-regulated genes.
• Metabolomic signature analysis in ‘secretome’ (in progress)
• Integration of existing data streams to explore correlations (in progress)
18
Activation of liver nuclear receptors Expanding (changing) the map of nuclear reception activation for PPAR-a
Slide Courtesy Mel Andersen, Hamner Institute for Chemical Safety Sciences
September 30 2014
Questions??
19
Thank You!!
September 30, 2014