Clinical Pathology - Theses

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Subject: Clustered regularly interspaced short palindromic repeats (CRISPR)-based strategy × Subject: dCas9-DNMT3A × Subject: epigenetic therapeutics × Type: PhD thesis ×

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    Heritable DNA methylation marks associated with familial breast and prostate cancer
    Hosseinpour, Mahnaz ( 2022)
    The known genetic risk factors for breast and prostate cancer account for less than 50% and 35% of the familial risk respectively. Early candidate gene studies provided evidence that DNA methylation measured in peripheral blood can mimic the effect of cancer predisposition associated with germline pathogenic variants. Key examples included the increased risk of breast cancer associated with both BRCA1 promoter hypermethylation and intragenic DNA methylation in ATM. More recently, the association between heritable DNA methylation marks and breast and/or prostate cancer risk has been reported by genome-wide studies. However, it is not clear how most of these modifications in DNA methylation mediate phenotypes resulting in increased cancer risk. In this thesis, a recently developed analytic approach, based on complex segregation analysis and family structure, was applied to identify 1,000 heritable DNA methylation marks in 57 families with multiple cases of breast and prostate cancer. Using Cox segregation analysis, we found that 229 and 140 methylation marks were associated with breast and prostate cancer respectively and 63 were associated with increased risk of both cancer types. We developed a Clustered regularly interspaced short palindromic repeats (CRISPR)-based strategy using a dCas9-DNA methyltransferase enzyme (DNMT) and a modified sgRNA containing two PP7 hairpins fused with DNMT3A in the synergistic activation mediator (SAM) system to assess whether induction of DNA methylation at these methylation sites resulted in a recognised cancer phenotype (such as DNA damage and proliferation). We first optimised CRISPR mediated DNA methylation by testing three combinations, including fusion of dCas9-DNMT3A, dCas9-DNMT3B and dCas9-DNMT1 with our modified sgRNA and found that SAM-DNMT3A which involves fusion of dCas9-DNMT3A with our modified sgRNA induced highly robust DNA methylation even compared to the previously developed approach, SunTag system, via transfection of HEK293FT cells targeting BRCA1 promoter region. In addition, we performed lentiviral transduction of human mammary breast cell lines using SAM-DNMT3A and sgRNAs targeting BRCA1, PTEN and NF1 regions and demonstrated a significant DNA methylation induction at these regions compared to GFP- sgRNA, targeting non-human genes. We found that the SAM-DNMT3A could also induce higher gene silencing in comparison with CRISPR-inhibition (CRISPRi) tool, involving dCas9-KRAB. A lentiviral delivered sgRNA pooled library, including 10 sgRNAs for each of the 1,000 heritable DNA methylation sites, was generated to perform phenotypic screens in the human mammary epithelial cells, B80-T5 and K5+/K19+ cells using SAM-DNNT3A and CRISPRi tools.We measured the effect of DNA methylation on proliferation and the DNA damage response using a PARP inhibitor synthetic lethal screen. K5+/K19+ cells, showed very low phenotypic changes. Overall, the SAM-DNMT3A tool enabled a systemic high throughput pooled screen of heritable DNA methylation sites mediating breast cancer related phenotypes including proliferation and DNA damage. The findings of this study further characterise the non-genetic component of familial risk of breast and prostate cancer, provides new opportunities to elaborate how tumorigenesis can be affected by DNA methylation and develop epigenetic therapeutics targeting these risk factors, ultimately advancing both precision prevention and medicine.