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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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Multi-omics analysis in GeneSpring Pathway-centric approach to multi-omics research powered by GeneSpring Analytics

September 30, 2014

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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?

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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

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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.

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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

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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)

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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??

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Thank You!!

September 30, 2014