Candidate Compounds for the Chemoprevention of Hereditary Diffuse Gastric Cancer

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline mutations in the tumour suppressor gene CDH1, which encodes the cell-cell adhesion protein E-cadherin. Mutations in CDH1 lead to increased proliferation, invasiveness and loss of cell polarity. Patients with germline CDH1 mutations have a 70% lifetime risk of developing gastric cancer. Female carriers also have a 42% lifetime risk of developing lobular breast cancer. The current optimal treatment for HDGC is a prophylactic gastrectomy as surveillance through endoscopy is imperfect and tumours can be discovered at a late stage. This project aims to utilise the synthetic lethal (SL) approach to find a chemopreventative treatment for HDGC. The SL approach aims to manipulate cell viability by exploiting the relationships between related genes. Synthetic lethality is a gene relationship in which an inactivation of either of two genes alone allows cell survival, however when both are inactivated it leads to cell death. Previous work within the laboratory has identified potential SL targets for CDH1 via high throughput drug screening. This was carried out in an isogenic pair of non-tumourigenic breast epithelial MCF-10A cell lines, one lacking CDH1 function. This identified histone de-acetylase inhibitors (HDACi) and statins as promising SL therapies for HDGC as they can selectively inhibit growth of CDH1-/- cells. Epigenetic changes, such as histone acetylation and deacetylation, modify expression patterns and are linked to cancer progression. Statins are widely used, well tolerated drugs that lower cholesterol levels and impact on plasma membrane organisation. This project has validated successful SL compounds from the MCF-10A drug screen in a gastric cancer model, using isogenic NCI-N87 CDH1+/+ and CDH1-/- cell lines. Promising compounds that have shown the best SL effect in 2D cell culture models, were then analysed for clinical trial data to find drugs that have minimal adverse effects. The compounds with minimal side effects and strong SL relationship with CDH1, were then assessed in a gastric organoid model of HDGC. This organoid model called the air-liquid interface (ALI) model, is a 3D representation of the gastric gland and contains all gastric cell lineages. As such it is a more accurate pre-clinical model for drug screening than 2D cultures. A second organoid model, the submerged model, was also further developed as it is a higher throughput model and will be more efficient for future drug compound testing. Mocetinostat, the compound identified to have the best synthetic lethal effect, will now undergo further preclinical testing as a potential chemopreventative compound for families affected by HDGC

Candidate Compounds for the Chemoprevention of Hereditary Diffuse Gastric Cancer

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline mutations in the tumour suppressor gene CDH1, which encodes the cell-cell adhesion protein E-cadherin. Mutations in CDH1 lead to increased proliferation, invasiveness and loss of cell polarity. Patients with germline CDH1 mutations have a 70% lifetime risk of developing gastric cancer. Female carriers also have a 42% lifetime risk of developing lobular breast cancer. The current optimal treatment for HDGC is a prophylactic gastrectomy as surveillance through endoscopy is imperfect and tumours can be discovered at a late stage. This project aims to utilise the synthetic lethal (SL) approach to find a chemopreventative treatment for HDGC. The SL approach aims to manipulate cell viability by exploiting the relationships between related genes. Synthetic lethality is a gene relationship in which an inactivation of either of two genes alone allows cell survival, however when both are inactivated it leads to cell death. Previous work within the laboratory has identified potential SL targets for CDH1 via high throughput drug screening. This was carried out in an isogenic pair of non-tumourigenic breast epithelial MCF-10A cell lines, one lacking CDH1 function. This identified histone de-acetylase inhibitors (HDACi) and statins as promising SL therapies for HDGC as they can selectively inhibit growth of CDH1-/- cells. Epigenetic changes, such as histone acetylation and deacetylation, modify expression patterns and are linked to cancer progression. Statins are widely used, well tolerated drugs that lower cholesterol levels and impact on plasma membrane organisation. This project has validated successful SL compounds from the MCF-10A drug screen in a gastric cancer model, using isogenic NCI-N87 CDH1+/+ and CDH1-/- cell lines. Promising compounds that have shown the best SL effect in 2D cell culture models, were then analysed for clinical trial data to find drugs that have minimal adverse effects. The compounds with minimal side effects and strong SL relationship with CDH1, were then assessed in a gastric organoid model of HDGC. This organoid model called the air-liquid interface (ALI) model, is a 3D representation of the gastric gland and contains all gastric cell lineages. As such it is a more accurate pre-clinical model for drug screening than 2D cultures. A second organoid model, the submerged model, was also further developed as it is a higher throughput model and will be more efficient for future drug compound testing. Mocetinostat, the compound identified to have the best synthetic lethal effect, will now undergo further preclinical testing as a potential chemopreventative compound for families affected by HDGC

Loss of E-Cadherin Leads to Druggable Vulnerabilities in Sphingolipid Metabolism and Vesicle Trafficking

Germline inactivating variants of CDH1 are causative of hereditary diffuse gastric cancer (HDGC), a cancer syndrome characterized by an increased risk of both diffuse gastric cancer and lobular breast cancer. Because loss of function mutations are difficult to target therapeutically, we have taken a synthetic lethal approach to identify targetable vulnerabilities in CDH1-null cells. We have previously observed that CDH1-null MCF10A cells exhibit a reduced rate of endocytosis relative to wildtype MCF10A cells. To determine whether this deficiency is associated with wider vulnerabilities in vesicle trafficking, we screened isogenic MCF10A cell lines with known inhibitors of autophagy, endocytosis, and sphingolipid metabolism. Relative to wildtype MCF10A cells, CDH1−/− MCF10A cells showed significantly greater sensitivity to several drugs targeting these processes, including the autophagy inhibitor chloroquine, the endocytosis inhibitors chlorpromazine and PP1, and the sphingosine kinase 1 inhibitor PF-543. Synthetic lethality was confirmed in both gastric and mammary organoid models of CDH1 loss, derived from CD44-Cre/Cdh1fl/fl/tdTomato mice. Collectively, these results suggest that both sphingolipid metabolism and vesicle trafficking represent previously unrecognised druggable vulnerabilities in CDH1-null cells and may lead to the development of new therapies for HDGC

E-Cadherin-Deficient Epithelial Cells Are Sensitive to HDAC Inhibitors

Inactivating germline mutations in the CDH1 gene (encoding the E-cadherin protein) are the genetic hallmark of hereditary diffuse gastric cancer (HDGC), and somatic CDH1 mutations are an early event in the development of sporadic diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In this study, histone deacetylase (HDAC) inhibitors were tested for their ability to preferentially inhibit the growth of human cell lines (MCF10A and NCI-N87) and murine organoids lacking CDH1 expression. CDH1−/− breast and gastric cells were more sensitive to the pan-HDAC inhibitors entinostat, pracinostat, mocetinostat and vorinostat than wild-type cells, with an elevated growth inhibition that was, in part, attributable to increased apoptosis. CDH1-null cells were also sensitive to more class-specific HDAC inhibitors, but compared to the pan-inhibitors, these effects were less robust to genetic background. Increased sensitivity to entinostat was also observed in gastric organoids with both Cdh1 and Tp53 deletions. However, the deletion of Tp53 largely abrogated the sensitivity of the Cdh1-null organoids to pracinostat and mocetinostat. Finally, entinostat enhanced Cdh1 expression in heterozygous Cdh1+/− murine organoids. In conclusion, entinostat is a promising drug for the chemoprevention and/or treatment of HDGC and may also be beneficial for the treatment of sporadic CDH1-deficient cancers

Hereditary diffuse gastric cancer: updated clinical practice guidelines

Hereditary diffuse gastric cancer (HDGC) is an autosomal dominant cancer syndrome that is characterised by a high prevalence of diffuse gastric cancer and lobular breast cancer. It is largely caused by inactivating germline mutations in the tumour suppressor gene CDH1, although pathogenic variants in CTNNA1 occur in a minority of families with HDGC. In this Policy Review, we present updated clinical practice guidelines for HDGC from the International Gastric Cancer Linkage Consortium (IGCLC), which recognise the emerging evidence of variability in gastric cancer risk between families with HDGC, the growing capability of endoscopic and histological surveillance in HDGC, and increased experience of managing long-term sequelae of total gastrectomy in young patients. To redress the balance between the accessibility, cost, and acceptance of genetic testing and the increased identification of pathogenic variant carriers, the HDGC genetic testing criteria have been relaxed, mainly through less restrictive age limits. Prophylactic total gastrectomy remains the recommended option for gastric cancer risk management in pathogenic CDH1 variant carriers. However, there is increasing confidence from the IGCLC that endoscopic surveillance in expert centres can be safely offered to patients who wish to postpone surgery, or to those whose risk of developing gastric cancer is not well defined.No Stomach for Cancer DeGregorio Foundation DD & DF Heads Charitable Trust University of Otago New Zealand Health Research Council Programme 17/610 Portuguese Foundation for Science and Technology POCI-01-0145-FEDER-3016