Progress and Future Directions in Integrated Systems Toxicology. Mary McBride Agilent Technologies

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1 Progress and Future Directions in Integrated Systems Toxicology Mary McBride Agilent Technologies 1

Toxicity testing tools of the late 20 th century Patchwork

in Animals with Extrapolation to Human-Relevant Doses

use of animals Large number of endpoints Not amenable to high

2 Toxicity testing tools of the late 20 th century Patchwork approach to testing dates back to the 1930 s 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 2

Toxicity testing in the 21 st century NRC report provided a vision and a strategy for shift to in vitro testing Report Called for a Transformative Change to Toxicity Testing, with 4 Major Components:

3 Toxicity testing in the 21 st century NRC report provided a vision and a strategy for shift to in vitro testing 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) NRC Report, 2007 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 vitroto-in vivo extrapolations 3

TT21C in practice: Systems toxicology approach Can provide deep mechanistic understanding of toxicological effects, enable prediction Systems Toxicology is: the integration of the classic toxicology

4 TT21C in practice: Systems toxicology approach Can provide deep mechanistic understanding of toxicological effects, enable prediction 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. Sturla, et.al. Systems Toxicology: from basic research to risk assessment. Chem. Res. Toxicol., 27: (PMID: ). 4

Systems biology to map and model pathways Using known toxicity pathways to

pathway assays In-vitro to in-vivo extrapolation xxx Compare to in-vivo outcomes

Computational Systems Biology Pathway Model Validated Pathway Assay Other data

5 Systems biology to map and model pathways Using known toxicity pathways to rapidly go from assays to risk assessments (Stem) cell biology, high-throughput pathway assays In-vitro to in-vivo extrapolation xxx Compare to in-vivo outcomes from MOA studies Multi-omics pathway analysis Time course, dose response Computational Systems Biology Pathway Model Validated Pathway Assay Other data streams: Ca transients, cell imaging, etc Computational cell biology, control theory, systems dynamics 5

Cell-based assays for predictive toxicology In-vitro platforms for

Primary Human Cells Limited availability Variable quality

Transformed Cell Lines May not recapitulate cell/tissue biology May

diversity Animal Models Human Stem Cells Represent human biology?

Cardiomyocytes Proliferate extensively Differentiate into any cell

development windows Enable access to population-level phenotypes

6 Cell-based assays for predictive toxicology In-vitro platforms for drug and chemical safety, toxicity testing and disease modeling Primary Human Cells Limited availability Variable quality Phenotypic instability Donor variability Limited characterization Transformed Cell Lines May not recapitulate cell/tissue biology May lack key functional characteristics Inadequately represents human diversity Animal Models Human Stem Cells Represent human biology? Resource intensive Animal welfare issues Chondrocytes Hepatocytes Cardiomyocytes 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 6

3D culture simulates in-vivo cell environment Innovations in 3D culture are enabling more

Cells partially interact Not representative of in vivo environment Porous, flexible (ECM

environment with micro-engineered controls DARPA/FDA/NIH Human-on-a-Chip Integrating organ

7 3D culture simulates in-vivo cell environment Innovations in 3D culture are enabling more biologically-relevant results 2D Cell Culture 2D 3D 3D Cell Culture 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 DARPA/FDA/NIH Human-on-a-Chip Integrating organ on a chip microdevices to produce physiologically and pathologically accurate models of human organ systems 7

View/quantify cellular dynamics in real time Advances in auto-focusing, sample positioning, SW

8 Cell-based imaging Enables visualization of molecular, biochemical, and cellular processes in living cells 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 8

Next-gen tailored in-vitro fit-for-purpose assays Need in-vitro assays to map

Validated Toxicity Pathway Assays CSBP Models in Vitro PK EC 10 P r + L P i -

AhR-Pathway Safety Assessment Reporter assay e.g.

Various high throughput tests Move on to specific in vitro pathway assays for

9 Next-gen tailored in-vitro fit-for-purpose assays Need in-vitro assays to map pathway circuitry, understand perturbations and MOAs Evaluate Results Validated Toxicity Pathway Assays CSBP Models in Vitro PK EC 10 P r + L P i - L Determine consistency of observed MOAs and expected targets E2-Pathway AhR-Pathway Safety Assessment Reporter assay e.g., most likely reproductive toxicity through E2 activation DNA-damage Pathway Various high throughput tests Move on to specific in vitro pathway assays for E2-pathway Oxidative Stress Pathway Mitochondrial Damage Pathways Slide courtesy Mel Andersen, Hamner Institute,

Automated high-throughput screening Tox21C has demonstrated rapid screening of in-vitro assays Each compound tested against battery of >700 biochemical and

, nuclear receptor, oxidative stress) Miniaturized assay volumes 2-6 ul in 1536-well plate Informatics pipeline for data processing, curve fitting & classification,

10 Automated high-throughput screening Tox21C has demonstrated rapid screening of in-vitro assays 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 10

11 Multi-omics pathway analysis of dose-response Illuminating biological understanding through a systems biology approach Genomics Transcriptomics Proteomics Metabolomics Genes mrna Protein Metabolite Production Regulation Prevailing paradigm for biological information flow does not fully describe the system 11

12 Multi-omics analysis in GeneSpring Pathway-centric approach to multi-omics research powered by GeneSpring Analytics 12

13 GeneSpring Metadata Framework (Ver. 15.xx) Visualize individual samples with their metadata information Can we unambiguously link adverse outcome phenotype to underlying MOA? 13

A private sector partnership for toxicity testing Partnership is accelerating implementation of in vitro tox testing Industry Technology Leverage $3M investments already made for Hamner

14 A private sector partnership for toxicity testing Partnership is accelerating implementation of in vitro tox testing Industry Technology Leverage $3M investments already made for Hamner estrogen-signaling case study approach; technology sector investments to extend/accelerate R&D Provide instrumentation and expertise for genomic, metabolomic, proteomic, and transcriptomic studies, bioinformatics, data analysis and visualization tools Governance Board One representative from each partner Development and oversight of research strategy and partner interactions Administrative budgeting, organization, etc. EPA- Regulatory/policy, risk assessment, computational models and database hosting. NIH, FDA may be better positioned to join in Academic Thought Leaders Direct and coordinate laboratory R&D. Develop assays and protocols. Analyze data. Work with public and private partner in developing software and bioinformatics tools. 14

15 Case studies using prototype pathways Using known toxicity pathways to rapidly go from assays to risk assessments 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. 15

16 A systems toxicology approach to PPAR-a pathway Developing a testing strategy for nuclear-receptor mediated proliferation 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 computational systems biology pathway (CSBP) models for Evaluate transcription factor network predicting acceptable exposures Examine functional relationships of regulated genes Measure metabolomic signatures in secretome 16

17 Activation of liver nuclear receptors Expanding the map of nuclear reception activation for PPAR-a 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) Slide Courtesy Mel Andersen, Hamner Institute for Chemical Safety Sciences 17

for PPAR-a Slide Courtesy Mel Andersen, Hamner

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 18 September

19 Questions?? Thank You!! 19

Tox21: Opportunities& Challenges. Richard A. Becker Ph.D., DABT American Chemistry Council

Tox21: Opportunities& Challenges Richard A. Becker Ph.D., DABT American Chemistry Council October 21, 2010 Opportunities and Challenges The manifest challenge is to develop in vitro and in silico approaches