Translating Genomes | Personalizing Medicine
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Using cell-based assays in the development of efficacious cancer therapies
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Translating Genomes | Personalizing Medicine
- 1. Translating Genomes | Personalizing Medicine Using cell-based assays in the development of efficacious cancer therapies Dr. Kyla Grimshaw VP Research Operations
- 2. 2 Presenter Kyla Grimshaw PhD VP 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.
- 3. 3 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
- 4. Translating genetic information into personalised medicine Genomics Translational Genomics Personalised Medicine 4
- 5. Horizon is a Products & Services Company 5
- 6. Genome Editing: To create accurate genetic models Isakoff et al., Cancer Research, Jan 2006 Di Nicolantonio et al., PNAS, Dec. 2008 6 Large growth induction phenotype Transforming alone Milder growth induction phenotype Non-transforming alone
- 7. 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, 7 new trials on-going In vitro cell models In vivo xenograft models Patient clinical trials
- 8. Horizon Discovery Research Division: Horizon’s services and collaboration platforms 8 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
- 9. Horizon Discovery Research Division: Horizon’s services and collaboration platforms 9 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 CombinatoRx platform In vivo platform Plus all functional assays typical of oncology drug discovery programmes
- 10. 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?
- 11. 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 900 800 700 600 500 400 300 200 100 0 HCT116 WT HCT116 KO 0 6 12 18 24 30 11 Knock-out of gene, in vivo Tumour Volume (mm3) Time (days) Knock-in of activity dead mutation, in 3D
- 12. Target Validation: A new Target Essentiality Analysis Infect cells targeting gene of interest Cas9 nuclease activity will introduce random double strand breaks into each allele of the target cell line. Culture colonies from single cells Assess length of fluorescent PCR products to check allele ratios and frame shift occurrence in 100’s colonies TARGET ESSENTIAL Colonies contain only in frame indels TARGET NON-ESSENTIAL Colonies contain frame shift indels
- 13. 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?
- 14. 14 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
- 15. Patient stratification: Isogenic cell line panel screening - genotype ID 15
- 16. 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 16 siRNA target ID Synthetic lethality waterfall plot (HCT116 isogenics) Hits with selective death in the mutant line
- 17. 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?
- 18. Resistance to a tyrosine kinase inhibitor is conferred by PTEN deletion or activating mutations of PIK3CA in colon cancer cells Cell viability
- 19. 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?
- 20. Effect of assay format on Olaparib response in DLD-1 colorectal cancer cells 96 h proliferation assay 10 day colony formation assay
- 21. Effect of oxygen levels on Tirapazamine response in HCT116 cells 3D spheroid Pimonidazole staining of spheroid cross-section
- 22. 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?
- 23. 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 Response to MEK inhibitors in 2D vs. 3D Log [M] ARRY162 (MEK inhibitor)
- 24. 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
- 25. High throughput cell-based drug combination assays Diverse Mutational Backgrounds Compounds Compounds Function 1 Function 2 Function 3 Function 4 Function 5 Function 6 Function 7 Function 8 Function 9 Function 10 Function 11 Function 12 Function 13 Function 14 Function 15 Function 16 Function 17 Function 18 Function 19 Function 20 Function 21 Function 22 Function 23 Function 24 Function 25 Function 26 Function 1 Function 2 Function 3 Function 4 Function 5 Function 6 Function 7 Function 8 Function 9 Function 10 Function 11 Function 12 Function 13 Function 14 Function 15 Function 16 Function 17 Function 18 Function 19 Function 20 Function 21 Function 22 Function 23 Function 24 Function 25 Function 26 Over 800 cell lines 250 enhancer agents Over a decade of experience in drug combination screening Measured Activities Excess
- 26. Horizon Discovery: Integrated Solutions Single agent 100-400 cancer cell lines Combination study 100-400 cancer cell lines 100+ enhancers HIT NOMINATION Isogenic Cell Lines Model generation AAV/CRISPR/ZFN PDX models ex-vivo, in vivo Highly characterised 26 Secondary assays Microenvironment, scheduling, higher order combinations MOA interrogation siRNA/sgRNA/shRNA Biomarkers assessment Isogenic panels Gene expression analysis For MOA insights and PD marker direction In vivo KO models Genetic engineering in rat or mouse models ZFN/CRISPR Xenograft models Standard mouse xenografts Bioinformatics Analysis Response predictors, clustering Isogenic cell lines Model generation, screening in directed panels
- 27. Recent New Developments: New Assay Models HaloTag® NanoLuc™ 27 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
- 28. Recent New Developments: New Assay Models 28 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 gut Growing mid/hind gut spheroids into human intestinal organoids
- 29. 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) 29 Shalem et al Science 2014
- 30. Repositioning Patient stratification Working with Horizon Services Division Target ID Target Validation H2L LO Generation of isogenic cell lines MOA assays to support med chem siRNA screens sgRNA screens TIDVAL alliances KO to test essentiality Activity dead KI mutations Compound profiling in isogenic cells Compound profiling in large Combination assays cell line panels Target validation & early stage drug discovery collaborations Finding a development path for stranded clinical assets In vivo models 30 Discovery Research Services Custom Cell Line Development CombinatoRx Custom Screening Services In vivo models
- 31. Your Horizon Contact: Dr. Kyla Grimshaw VP Research Operations k.grimshaw@horizondiscovery.com +44 (0)1223 655580 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: info@horizondiscovery.com Web: www.horizondiscovery.com
Notes de l'éditeur
- Welcome and thank you for joining me today to talk about cell-based assays at Horizon Discovery. In the webinar today I would like to introduce you to Horizon Discovery and describe our functional assays division Focussing on how we use cell based assays to aid our customers and collaborators to perform first class oncology drug discovery.
- Information is no longer a bottleneck, emphasis is shifting to the ‘what does it all mean’ Genome editing is enabling the promise of the genomic era to be realized in the form of novel therapeutics and diagnostics It involves the capability to efficiently introduce targeted alterations into any specific gene in living cells
- Horizon offers a range of products & services to support this discovery pipeline, from basic research through to validated therapeutic targets. At Horizon, cell line models can be accessed by multiple routes to suit your requirements. Previously created isogenic cell lines can be accessed via our range of of-the-shelf cell lines, or else we can provide reagents and design advice via the GenAssist service through to a full custom cell line development project, generating your specific mutation in the cell line of your choice, performed by Horizons experienced scientists. Target discovery and drug development services now include our high throughput CombinatoRx platform, and in vivo models at our Sage labs facility. These services can be accessed individually or in as an integrated project to enable us to support projects of any size.
- Horizon is founded on the use of genome editing, and this is fundamental to our philosophy supporting oncology drug discovery. And here’s why: Historically, to understand the effect of a specific mutation, classic overexpression studies have been performed, in which a mutated gene of interest is introduced randomly into the cell under the control of an exogenous promoter. This can result in unclear results due to the non-physiological levels of expression and regulation. For example, on the slides we can see two studies investigating the effect of the same mutations. The first overexpresses PI3Kinase mutations and resulted in an oncogenic phenotype. However, when these same mutations are introduced into the endogenous PI3K gene, where physiological levels of expression and endogenous mechanisms of regulation are retained, a much milder growth phenotype is observed which alone would not be considered as transforming. This more accurate modelling of the impact of specific mutations enables the multifactoral genotypes present if complex disease to be investigated with greater confidence. Similar limitations can be seen with si and sh RNA studies which attempt to knock-down the expression of genes of interest. However these are rarely complete knockdowns and the residual expression cannot be discounted. Whereas permanent, stable disruption of a gene removes such confounding issues.
- We have devised a medium-throughput method that can shed light on the ambiguous results that emerge from siRNA/shRNA or sgRNA screens shRNA only gives partial knockdown; growth phenotypes often partial too Repair of Cas9-mediated ds breaks can result in in-frame indels that don’t disrupt protein function We would use lentiviruses to deliver Cas9 + sgRNA vs target to cells, allow 14-20 days for gene editing to occur and then culture colonies from single cells Horizon’s cell-line engineering experience allows us to devise an analysis pipeline to characterise editing events on a clonal level
- Three technical replicates per screen (CVs liquid handling <2.5%; CVs +/- controls ~ 13%) Blue line is the median parental response plotted as a 2D waterfall Green data points are the mutant response for the matching target gene
- 96h reveals some sensitivity to BRACS null lines. 10 day colony forming assay definitively demonstrates.
- In a 2D cell culture assay tirapazamine requires exposure to low oxygen conditions for activation and to exert anti-proliferative effects. Activity is seen in a 3D spheroid model which contains a hypoxic core Cell line?
- Finally, An exciting new area that is gaining a lot of interest at the moment are endogenous reporter lines. The same limitations that apply to over-expression studies apply to systems employigg synthetic reporter systems. In collaboration with Promega we have generated panels of cell lines that contain genes endogenously tagged with either the multi-purpose Halo-Tag or Nano Luc – an extremely bright reporter, over 150x as bright as standard firefly luciferase using this technology, it is now possible to monitor the regulation of gene expression at the endogenous level and are sutiable for use in high-throughput formats.
- And we are also investing in more complex disease models in the form of both PDX and organoids. Though Sage labs we have developed 25 breast cancer PDX models. These are one of the most characterised sources of breast PDX models in the world, and are currently being established in our labs. And we are also very interested to explore the possibilities of growing iPS organoids, both for screening and cell line engineering purposes .The data shown here is from our labs where we are at the exciting stage of sectioning fully formed organoids.
- And if you would like to hear more about this exciting new area, please register for our sgRNA screening webinar n the 9th Dec. Cancer is a genetic disease Genome sequencing has generated 100’s of potential targets for cancer therapy However, most targets are rare and poorly characterised Most of them can’t be drugged directly e.g., tumour suppressors If tumour suppressor loss is the prime cancer initiating event, agents exploiting this loss may assist with overcoming tumour heterogeneity Systematic de-orphaning required to find key druggable downstream targets - exploiting co-dependence or synthetic lethality
- Additional slide options