Functional validation of cancer susceptibility genes using gene editing
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1 Functional validation of cancer susceptibility genes using gene editing Sabine Topka Research Fellow Niehaus Center for Inherited Cancer Genomics
2 Inherited Predisposition to Cancer Hereditary mutations account for 5-10% of all cancers High penetrance, rare cancer predisposition genes (Relative risk 5) Discovery of novel cancer predisposition genes in humans Linkage studies in cancer-prone kindreds Genome wide association studies (GWAS) Next generation sequencing (NGS) Inherited variation and cancer susceptibility High-penetrance mutations (causative in isolation, but rare) Moderate-penetrance mutations (more common, interact with other genetic and nongenetic factors to influence an individual's cancer risk) Low penetrance genomic variants (Common, modifying effect on cancer risk) Weitzel et al, CA: A Cancer Journal for Clinicians, 61(2011),
3 Germline Susceptibility in Breast Cancer Highly heritability of breast cancer (~15% of cases with family history) Half of the familial aggregation for breast cancer remains unexplained Couch et al, Science, 343(2014),
4 Importance of precise model systems for functional validation of cancer susceptibility genes Risk assessment Precise evaluation of risk is crucial for counseling patients that are diagnosed with a germline mutation Limited availability of patient cell lines Need for precise model system (haploinsuffic iency) CRISPR/Cas9 genome editing Treatment optimization (targeted therapy) Genetic counseling of patients Decission about prophylactic measures Affects decisions about prophylactic measures such as screening/medical decisions and family planning Treatment optimization (targeted therapy) and avoidance of risk (eg., reduce/avoid irradiation exposure) Often mutations act as haploinsufficient and thus cannot be adequately modeled by overexpression systems Cell lines from patients are often not easily available especially not from tissue of interest CRISPR/Cas9 genome editing has the potential to quickly and precisely model mutations and asses their functional effects
5 Identification of a Recurrent ERCC3 R109X Mutation in Ashkenazi Probands with BRCA1/2 Negative Breast Cancer Exome sequencing of 46 early onset (<45 years) and 13 familial BRCA wild type breast cancer probands ERCC3 R109X mutation identified in 5 individuals Case-control association study (>3000 cases & controls) estimates >1.5x increased breast cancer risk for carriers Haplotype length suggests founder mutation! Vijai, Topka et al, Cancer Discovery, 6(2016),
6 ERCC3/XPB and nucleotide excision repair ATP-dependent 3'-5' DNA helicase Component of the core-tfiih basal transcription factor Involved in RNA transcription by RNA polymerase II and in nucleotide excision repair (NER) Acts by opening DNA either around the RNA transcription start site or the DNA damage Kamilieri et al, Trends in Genetics, 28(2012),
7 Sequencing of ERCC3 patient gdna/cdna WT c.325c>t gdna c.325c>t cdna Lower expression of mutated allele in patient sample Vijai, Topka et al, Cancer Discovery, 6(2016),
8 Creation of ERCC3 Mutants in Human Mammary Epithelial Cells by Genome Editing Reduced ERCC3 mrna and protein levels in R109X CRISPR models Vijai, Topka et al, Cancer Discovery, 6(2016),
9 Functional Characterization of Genome Edited ERCC3 Mutants in Human Mammary Epithelial Cells ERCC3 mutant cell lines show increased sensitivity against alkylating DNA damage induced by fungal sesquiterpene IlludinS Vijai, Topka et al, Cancer Discovery, 6(2016),
10 Functional Characterization of Genome Edited ERCC3 Mutants in Human Mammary Epithelial Cells Removal of H2AX phosphorylation following DNA damage suggests impaired repair kinetics in ERCC3 mutant cell lines Vijai, Topka et al, Cancer Discovery, 6(2016),
11 Common strategies to increase editing and HDR efficiencies guiderna design and testing: optimal location and cutting efficiency T7 Endonuclease Repair donor design considerations: optimal length of homology arms directionality/asymmetry Increasing rate of HDR: increase number of cells that are in optimal cell cycle phase: synchronization (e.g., Nocodazole) use of NHEJ inhibitors Delivery method: Cas9-sgRNP complex nucleofection Selection of transfected cells co-transfection with GFP and sorting of transfected cells
12 How to find the needle in the haystack Screening method pre-screening of transfected cell pools using droplet digital PCR significantly reduces number of clones to screen Miyaoka et al, Nat Meth, 11(2014), 291-3
13 Droplet Digital PCR (ddpcr ) Technology Partitioning of samples (20µl) into 20,000 droplets each (Bio-Rad QX100 or QX200) PCR reaction within droplets read fluorescence of each droplet detection sensitivity as low as 1 in 5,000 (0.02%) Dynamic range within 3.3 fg to 330 ng per 20μl reaction (for hgdna)
14 HDR Edit Detection assay: two amplicon strategy HDR Edit Assay 2 amplicon Probe Binding HDR Edit Assay Duplex Readout TARGET ASSAY DARK (binds WT) FAM (Mutant) * WT HDR FAM+ = HDR Edit HEX+ = All Copies REFERENCE ASSAY HEX (Reference) WT + NHEJ + HDR Implications: Fractional abundance is given by the Ratio in Quantasoft All sequence structures have same readout with this assay
15 Advantages of Droplet Digital PCR High sensitivity allows for detection of very rare events Detection by T7 Endonuclease (NHEJ) or RFLP (HDR) quite insensitive RFLP Clonal Sequencing Faster turnaround time compared to Clonal Sanger Sequencing Lower cost and reduced workload than NGS NGS
16 ddpcr screen to identify HDR editing efficiency in transfected cell pools 4.8% 11% 6% 3.6% Neg ctrl Pos ctrl Expansion of pools with highest editing efficiency to generate higher number of modified single cell clones
17 Modeling of a BRCA2 founder mutation Brca2 WT/WT 1/116 (0.86%) HDR edited clone without pre-screening Brca2 WT/6174delT 4/57 (7%) HDR edited clones after ddpcr pre-screening Brca2 GAPDH Reduced transcript and protein levels in CRIPSR clones, Expression of truncated protein not detectable
18 Modeling of a BRCA2 founder mutation treatment with translation inhibitor restores normal mrna levels in heterozygous Brca2 mutant cells
19 Modeling of a BRCA2 founder mutation Brca2 mutant cell line shows higher sensitivity against platinum drug treatment
20 Towards multiplex CRISPR screening for functional validation of germline variants in cancer predisposition genes Identification of target regions and design of sgrnas and repair templates Functional screening and identification of potential pathogenic variants
21 Conclusions Inherited variants contribute to a significant proportion of cancer risk Identification and functional characterization of a novel DNA repair gene mutation associated with breast cancer Precise model system is needed for functional evaluation of cancer susceptibility genes CRISPR/Cas9 as an effective tool to interrogate genetic variants Improvements to precise genome editing have largely focused on increasing HDR editing rates Screening for HDR edited cells can be significantly improved by droplet digital PCR
22 Acknowledgements MSKCC Offit lab Kenneth Offit Vijai Joseph Vignesh Ravichandran Ann Maria Zarina Fnu igo core facility Daoqi You Ventura&Benezra labs Flow cytometry core facility Mark Tomishima (Stem Cell Core) Sharon Corzine Cancer Research Fund Robert and Kate Niehaus Center for Inherited Cancer Genomics Miele family initiative Andrew Sabin Family Fund NIH R01CA BCRF R21-A Bio-Rad Eli Hefner Jen Berman Samantha Cooper Madhuri Ganta Dianna Maar Bruce Cutson
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