A Synthetic Lethal Approach to the Development of Novel Cancer Therapies
The cell-cell adhesion protein E-cadherin (CDH1) is a tumour suppressor that is required to maintain cell adhesion, cell polarity and cell survival signalling. Somatic mutations in CDH1 are common in diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In addition, germline mutations in CDH1 predispose to the familial cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC).HDGC is characterised by multiple foci of stage T1a signet ring cell carcinomas (SRCC) that develop in the stomachs of CDH1 mutation carriers following the downregulation of the second CDH1 allele. Individuals from HDGC families have an ~70% lifetime risk of developing DGC. Females with germline CDH1 mutations have an additional ~40% lifetime risk of developing LBC.We have hypothesised that the loss of E-cadherin within early stage DGC and LBC could be specifically targeted using a synthetic lethal approach. A drug that targets the synthetic lethal partner of E-cadherin would be predicted to result in a lethal phenotype in cells lacking E-cadherin, but not wild-type cells. Novel compounds may provide an alternative approach to the prevention of DGC and LBC by eliminating cancer cells with inactivating CDH1 mutations before they have had an opportunity to progress.At present, prophylactic total gastrectomy is the only option available to eliminate an inherited risk of gastric cancer – this treatment is recommended for CDH1 mutation carriers from the age of 20 years. Unfortunately, most patients have significant morbidity post-gastrectomy and 27-36% of patients have major adverse events. Although these complications are gradually being better managed leading to better quality of life, even in younger patients, physical symptoms can persist and affect long-term quality of life. The breast cancer risk in HDGC families is usually managed by routine screening (annual magnetic resonance imaging); prophylactic mastectomy is currently not generally recommended, but remains an option for some women. Prophylactic mastectomies are however, common in women with lobular carcinoma in situ (LCIS). LCIS, which are frequently CDH1 negative, are associated with an 8-10 fold increased risk of LBC.We propose that E-cadherin synthetic lethal drugs will provide a treatment option for HDGC family members and women with LCIS that will reduce the need for prophylactic surgery.To identify novel synthetic lethal compounds for the treatment of cancers associated with E-cadherin loss, a high-throughput screening campaign of ~114,000 lead-like compounds was performed on an isogenic pair of human mammary epithelial cell lines – with and without CDH1 expression. This approach identified 84 compounds belonging to 13 distinct pharmacophore groups that were selectively lethal to E-cadherin-deficient cells. Validation of these groups using both real-time and end-point viability assays identified six novel compounds with significant synthetic lethal activity, thereby demonstrating that E-cadherin loss creates druggable vulnerabilities within tumour cells. Two of these lead compounds were then chosen to create a series of analogues to explore structure activity relationships with the purpose of determining an appropriate site for the attachment of a solid support for future target identification.Using CRISPR-Cas9 and Cas9-nickase, further isogenic cell lines with and without CDH1 expression were created for the validation of lead synthetic lethal compounds. Four sgRNA target sites against CDH1 were validated in MCF-7 cell lines. The CRISPR-Cas9 sgRNA site targeting CDH1 exon 10 was subsequently used to successfully edit MCF-7 and NCI-N87 polyclonal cells. However, attempts to create monoclonal cell lines with CRISPR induced CDH1 knock-outs were unsuccessful.In summary, we have developed a novel class of synthetic lethal compounds that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC.
Advisor: Guilford, Parry
Degree Name: Doctor of Philosophy
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Hereditary Diffuse Gastric Cancer; HDGC; E-cadherin; Lobular Breast Cancer; Synthetic Lethal; High-throughput Screen; CRISPR; MCF10A
Research Type: Thesis