Abstract
The only preventative treatment for carriers of genetic variants that cause hereditary diffuse gastric cancer (HDGC), a disease with poor prognosis, is total prophylactic gastrectomy.
Gastric cancer is the third most lethal and fifth most common cancer worldwide. Diffuse-type gastric cancer often develops due to inherited or somatic variants in the tumour suppressor gene E-cadherin (CDH1). Germline inactivation of CDH1 causes the inherited cancer syndrome HDGC, with up to 70% lifetime penetrance. Currently, the only treatment for this condition is total prophylactic gastrectomy, driving the search for an alternative. Dasatinib, an ATP-competitive tyrosine kinase inhibitor, targets pathways that are upregulated in CDH1-null cancer cells (primarily the SRC/AKT/DDR2 signalling axis). Consequently, it is considered a potential chemopreventative drug for carriers of CDH1 loss of function variants. To improve this targeted therapy, we aimed to identify synergistic partners of dasatinib, drugs which provide a greater than additive effect when combined with dasatinib. Synergistic drug combinations would enable reduced dasatinib dose and potentially reduced side effects, as well as reducing the risk of tumours developing resistance to a single drug.
Using a large, publicly available cancer gene expression database (the Cancer Cell Line Encyclopaedia), we compared differential gene expression patterns between cell lines that were susceptible and resistant to dasatinib. Reactome pathway over-representation analysis was carried out to identify pathways differentially enriched between these two groups. These were clustered based on their gene composition to create four larger dasatinib response signatures. We combined these signatures with drug sensitivity data (from the Genomics of Drug Sensitivity in Cancer database) to identify compounds whose efficacy correlated with signature activity, providing a list of candidate synergistic partners of dasatinib. Ten compounds were selected for validation based on the in silico results: acetalax, axitinib, AZD8055, camptothecin, gefitinib, sapitinib, SB216763, sorafenib, staurosporine, and zorifertinib. These targeted a variety of dasatinib-associated pathways, primarily those involved in extracellular matrix interactions and maintenance, along with PI3K/AKT/mTOR signalling and ERBB family activity.
We also took a second approach to identify drugs that were synergistic with dasatinib. A preliminary RNA-seq analysis of an HDGC model after dasatinib treatment revealed changes in pathway regulation in response to the drug. We followed this with a single-cell RNA-seq experiment to identify changes that occurred in response to dasatinib at a higher resolution, representing emerging mechanism of dasatinib resistance. We treated early stage diffuse gastric cancers in an inducible Cdh1-/-/Trp53-/- HDGC mouse model with dasatinib. Immediately following treatment, stomachs were resected for single-cell RNA sequencing, which enabled the identification of a distinct cluster of putative gastric cancer cells. Gene set enrichment analysis identified Reactome pathways that were altered between treatment and control groups, including RHO GTPase, MAPK, and YAP/TAZ signalling, oxidative phosphorylation, and autophagy. We selected five drugs that target these pathways for validation: defactinib, trametinib, verteporfin, metformin, and chloroquine.
To validate our bioinformatic approach to identifying drugs that were synergistic with dasatinib, the 15 drugs mentioned above were tested alone and in combination with dasatinib in preclinical gastric cancer models. Drug synergy was quantified using the Loewe additivity method. Gefitinib, AZD8055, and defactinib were found to be synergistic with dasatinib in both the NCI-N87 cell line and a murine gastric organoid model of HDGC. All three compounds (particularly gefitinib) exhibited greater synergy in CDH1-/- organoids compared to wild-type controls.
The identification of these combinations marks a significant step towards developing a durable and stable chemopreventative treatment for HDGC. Our advances in this field offer new hope to CDH1 variant carriers that they and their children may no longer have to contemplate life without a stomach.