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ObjectiveGastro-oesophageal cancers (GEC) are resistant to therapy and lead to poor prognosis. The cancer stem cells (CSCs) and antiapoptotic pathways often confer therapy resistance. We sought to elucidate the antitumour action of a BCL-2 inhibitor, AT101 in GEC in vitro, in vivo and in a clinical trial.MethodsExtensive preclinical studies in vitro and in vivo were carried out to establish the mechanism action of AT101 on targeting CSCs and antiapoptotic proteins. A pilot clinical trial in patients with GEC was completed with AT-101 added to standard chemoradiation.ResultsOverexpression of BCL-2 and MCL-1 was noted in gastric cancer tissues (GC). AT-101 induced apoptosis, reduced proliferation and tumour sphere formation in MCL-1/BCL-2 high GC cells. Interestingly, AT101 dramatically downregulated genes (YAP-1/Sox9) that control CSCs in GEC cell lines regardless of BCL-2/MCL-1 expression. Addition of docetaxel to AT-101 amplified its antiproliferation and induced apoptosis effects. In vivo studies confirmed the combination of AT101 and docetaxel demonstrated stronger antitumour activity accompanied with significant decrease of CSCs biomarkers (YAP1/SOX9). In a pilot clinical trial, 13 patients with oesophageal cancer (EC) received AT101 orally concurrently with chemoradiation. We observed dramatic clinical complete responses and encouraging overall survival in these patients. Clinical specimen analyses revealed that AT-101 dramatically reduced the expression of CSCs genes in treated EC specimens indicating antitumour activity of AT101 relies more on its anti-CSCs activity.ConclusionsOur preclinical and clinical data suggest that AT-101 overcomes resistance by targeting CSCs pathways suggesting a novel mechanism of action of AT101 in patients with GEC.

ObjectivePeritoneal carcinomatosis (PC; malignant ascites or implants) occurs in approximately 45% of advanced gastric adenocarcinoma (GAC) patients and associated with a poor survival. The molecular events leading to PC are unknown. The yes-associated protein 1 (YAP1) oncogene has emerged in many tumour types, but its clinical significance in PC is unclear. Here, we investigated the role of YAP1 in PC and its potential as a therapeutic target.MethodsPatient-derived PC cells, patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models were used to study the function of YAP1 in vitro and in vivo. Immunofluorescence and immunohistochemical staining, RNA sequencing (RNA-Seq) and single-cell RNA-Seq (sc-RNA-Seq) were used to elucidate the expression of YAP1 and PC cell heterogeneity. LentiCRISPR/Cas9 knockout of YAP1 and a YAP1 inhibitor were used to dissect its role in PC metastases.ResultsYAP1 was highly upregulated in PC tumour cells, conferred cancer stem cell (CSC) properties and appeared to be a metastatic driver. Dual staining of YAP1/EpCAM and sc-RNA-Seq revealed that PC tumour cells were highly heterogeneous, YAP1highPC cells had CSC-like properties and easily formed PDX/PDO tumours but also formed PC in mice, while genetic knockout YAP1 significantly slowed tumour growth and eliminated PC in PDO model. Additionally, pharmacologic inhibition of YAP1 specifically reduced CSC-like properties and suppressed tumour growth in YAP1highPC cells especially in combination with cytotoxics in vivo PDX model.ConclusionsYAP1 is essential for PC that is attenuated by YAP1 inhibition. Our data provide a strong rationale to target YAP1 in clinic for GAC patients with PC.

AbstractPanellus stypticusfruits widely in Northern Hemisphere temperate forests but has also been collected in Alaska, Costa Rica, Australia and New Zealand. Previous studies established that there was a single biological species across eastern North America, Russia, Japan and New Zealand. In order to examine phylogenetic and biogeographical patterns within this species, the ribosomal ITS1-5.8S-ITS2 region was amplified for 67 collections representing North America, Eurasia, Australia and New Zealand and was sequenced for ten exemplars selected from different geographical areas. The amplified ITS sequence length for collections from New South Wales and Tasmania (Australia) and New Zealand was consistent within each of these geographical areas but varied between areas and was shorter than the ITS region for Northern Hemisphere collections. Length changes were due to a number of deletions. Deleted regions totaled 15 bp in collections from New South Wales, 29 bp from Tasmania and 88 bp from New Zealand. Parsimony analysis based on ITS sequences produced two major clades, a Northern Hemisphere clade and an Oceanian clade. The collection from New South Wales was basal to the Oceanian clade with collections from Tasmania and New Zealand as derived. Restriction fragment length polymorphisms based on BstZI, DraI and HinfI identified several biogeographically separated haplotypes within this species. In the Northern Hemisphere, an eastern North American haplotype was separated from other P. stypticuscollections that extended from Pacific North America to Asia. European collections also shared a unique haplotype. The presence of a nuclear ribosomal large subunit group I intron was inferred in all Northern Hemisphere collections and in collections from New South Wales by length variation of the PCR product and by sequencing of selected exemplars. ITS sequence data and the presence of a group I intron in the Northern Hemisphere and New South Wales collections provide evidence that Northern Hemisphere and Southern Hemisphere distributions were connected through South East Asia.