Translating Genomes | Personalizing Medicine
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Using cell-based assays in the development of efficacious cancer therapies Translating Genomes | Personalizing Medicine Dr. Kyla Grimshaw VP Research Operations
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Using cell-based assays in the development of efficacious cancer therapies
Transcript of Translating Genomes | Personalizing Medicine
Using cell-based assays in the development of efficacious cancer therapies
Translating Genomes | Personalizing Medicine
Dr. Kyla GrimshawVP Research Operations
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Presenter
Kyla Grimshaw PhDVP Research Operations
Kyla has been leading the oncology translational research service at Horizon since co-founding the CRO arm of the company in 2007.
Prior to this, she worked on multiple drug discovery programmes at Millennium Pharmaceuticals and Astex Pharmaceuticals.
Her core area of expertise is in oncology cell biology and providing expert contract research to customers.
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Content of the Presentation
Introduction to Horizon Discovery
Horizon Discovery Research Division - Horizon’s services and collaboration platforms
Applying cell-based assays to address key questions in cancer drug discovery• Use of isogenic cell lines• Compound vs. genetic knockdown studies• Tumour microenvironment• Combination assays
New developments
Summary
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Translating genetic information into personalised medicine
Genomics Personalised MedicineTranslational Genomics
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Horizon is a Products & Services Company
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Genome Editing: To create accurate genetic models
Large growth induction phenotype
Transforming alone
Milder growth induction phenotype
Non-transforming alone
Di Nicolantonio et al., PNAS, Dec. 2008Isakoff et al., Cancer Research, Jan 2006
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Genome Editing: To translate in vitro discoveries to patient treatments
All K-RAS mutant patients excluded from therapy with Erbitux® EGFR targeted agent
X-MAN™ lines predicted Erbitux® may work for a 20% sub-set of these patients with G13D, new trials on-going
In vitro cell models In vivo xenograft models Patient clinical trials
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Horizon Discovery Research Division: Horizon’s services and collaboration platforms
Tool 1: X-MAN™ Cell Lines• Patient stratification• Target validation• Pathway interrogation
Tool 2: Tumour Microenvironment• 3D assays• Co-culture• Hypoxia, nutrient starvation
Tool 3: Screening Platforms• siRNA• shRNA• sgRNA
Plus all functional assays typical of oncology drug discovery programmes
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Horizon Discovery Research Division: Horizon’s services and collaboration platforms
Tool 1: X-MAN Cell Lines• Patient stratification• Target validation• Pathway interrogation
Tool 2: Tumour Microenvironment• 3D assays• Co-culture• Hypoxia, nutrient starvation
Tool 3: Screening Platforms• siRNA• shRNA• sgRNA
Plus all functional assays typical of oncology drug discovery programmes
CombinatoRx platform
In vivo platform
Key Questions: Cell-Based Assays in Cancer Drug and Target Discovery
Is the target well validated?
Which patients should I target?
Which patients should I avoid?
Is my assay configured correctly?
Impact of tumour microenvironment
What about combination therapy?
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HCT116 WT HCT116
KO
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Target Validation: Use of Knock-down, Knock-out and Knock-in experiments
shRNA target knock-down in A549 cells sgRNA target knock-out in A549 cells
Knock-out of gene, in vivo
Tum
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olu
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Time (days)
Knock-in of activity dead mutation, in 3D
Target Validation: A new Target Essentiality Analysis
Assess length of fluorescent PCRproducts to check allele ratios andframe shift occurrence in 100’scolonies
Culture colonies from single cells
Infect cells targeting gene of interest
TARGET ESSENTIALColonies contain only in
frame indels
TARGET NON-ESSENTIALColonies contain frame
shift indels
Cas9 nuclease activity will introducerandom double strand breaks intoeach allele of the target cell line.
Cell-Based Assays in Cancer Drug and Target Discovery
Is the target well validated?
Which patients should I target?
Which patients should I avoid?
Is my assay configured correctly?
Impact of tumour microenvironment
What about combination therapy?
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Isogenic cell lines
Horizon has collection of over 550 isogenic cell line pairs X-MAN™ cell lines
Horizon’s expert targeting team can also engineer a genetically validated cell line to a customer’s specification
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Patient stratification: Isogenic cell line panel screening - genotype ID
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Target identification using synthetic lethality screening and isogenic cell lines
Precisely control for target genotype and perfectly matched ‘normal’
Removes a key source of noise in synthetic lethality screens
siRNA target ID
Synthetic lethality waterfall plot (HCT116 isogenics)
Hits with selective death in the mutant line
Cell-Based Assays in Cancer Drug and Target Discovery
Is the target well validated?
Which patients should I target?
Which patients should I avoid?
Is my assay configured correctly?
Impact of tumour microenvironment
What about combination therapy?
Resistance to a tyrosine kinase inhibitor is conferred by PTEN deletion or activating mutations of PIK3CA in colon cancer cells
Cel
l via
bili
ty
Cell-Based Assays in Cancer Drug and Target Discovery
Is the target well validated?
Which patients should I target?
Which patients should I avoid?
Is my assay configured correctly?
Impact of tumour microenvironment
What about combination therapy?
Effect of assay format on Olaparib response in DLD-1 colorectal cancer cells
10 day colony formation assay96 h proliferation assay
Effect of oxygen levels on Tirapazamine response in HCT116 cells
Pimonidazole staining of spheroid cross-section
3D spheroid
Cell-Based Assays in Cancer Drug and Target Discovery
Is the target well validated?
Which patients should I target?
Which patients should I avoid?
Is my assay configured correctly?
Impact of tumour microenvironment
What about combination therapy?
3D conditions reveal a dependence on oncogenic KRAS in colon cancer cells
2D Adherent 3D Soft Agar
DLD-1 KRASG13D
DLD-1 KRASG13D KO
siRNA to KRAS in 3D
Log [M] ARRY162 (MEK inhibitor)
Response to MEK inhibitors in 2D vs. 3D
Cell-Based Assays in Cancer Drug and Target Discovery
Is the target well validated?
Which patients should I target?
Which patients should I avoid?
Is my assay configured correctly?
Impact of tumour microenvironment
What about combination therapy
High throughput cell-based drug combination assays
Compounds
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Over 800 cell lines
250 enhancer agents
Over a decade of experience in drug combination screening
Measured Activities Excess
PDX modelsex-vivo, in vivo
Highly characterised
Isogenic Cell Lines Model generationAAV/CRISPR/ZFN
Combination study100-400 cancer cell lines
100+ enhancers
Single agent100-400 cancer cell lines
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Horizon Discovery: Integrated Solutions
HIT NOMINATION
Secondary assaysMicroenvironment,
scheduling, higher order combinations
MOA interrogationsiRNA/sgRNA/shRNA
Biomarkers assessmentIsogenic panels
Gene expression analysisFor MOA insights and PD
marker direction
In vivo KO models Genetic engineering in rat or mouse models
ZFN/CRISPR
Xenograft modelsStandard mouse
xenografts
Bioinformatics AnalysisResponse predictors,
clustering
Isogenic cell linesModel generation,
screening in directed panels
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Recent New Developments: New Assay Models
Endogenous reporter cell lines and kits • X-MAN™ HaloTag® Reporter Kits Knock-in of reporters/tag into endogenous locus for in vitro imaging / pull-downs /
purification
• X-MAN™ NanoLuc™ Reporter Kits Knock-in of Luciferase reporter into endogenous locus for in vitro & in vivo pathway read-
outs
TMR ligand DNA Stain
HaloTag® NanoLuc™
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Recent New Developments: New Assay Models
PDX models• 25 PDX models of breast cancer, characterised by whole genome
sequencing• WGS has also been performed on the originating tumors, early and
late passages of PDX, as well as metastases, demonstrating striking similarity and faithful modelling of the original tumor by WHIM PDX models.
iPS organoids• Currently under development
Maintenance of undifferentiated iPS cells
Differentiation into human endoderm
Differentiation into human mid/hind gutGrowing mid/hind gut spheroids into human intestinal organoids
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Recent New Developments: Synthetic Lethal Target ID via sgRNA Screening
Webinar: RNA-based screening in drug discovery – introducing sgRNA technologies
Tue 9th Dec at 4 pm (GMT)
Shalem et al Science 2014
RepositioningPatient
stratificationLOH2LTarget ValidationTarget ID
siRNA screens
sgRNA screens
TIDVAL alliances
KO to test essentiality
Activity dead KI mutations
Generation of isogenic cell lines MOA assays to support med chemCompound profiling in
isogenic cells
Combination assays
Compound profiling in large cell line panels
Target validation & early stage drug discovery collaborations
Finding a development path for stranded clinical assets
In vivo models
Working with Horizon Services Division
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Discovery Research Services
Custom Cell Line Development
CombinatoRxCustom Screening
ServicesIn vivo models
Your Horizon Contact:
Horizon Discovery Ltd, Building 7100, Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 862 240 Email: [email protected] Web:
www.horizondiscovery.com
Dr. Kyla Grimshaw
VP Research Operations
+44 (0)1223 655580
