COLO 320 HSR

ecDNA Hubs Drive Cooperative Intermolecular Oncogene Expression

Extrachromosomal DNA (ecDNA) is prevalent in human cancers, where it contributes to oncogene overexpression by amplification and deregulation. Although gene activation is commonly explained by cis-regulatory elements on the same chromosome, the functional relevance of ecDNA clustering is not well understood. Here, Hung et al. explored how ecDNA hubs—clusters of 10–100 ecDNAs located in discrete nuclear bodies—facilitate intermolecular enhancer-gene communication and oncogene overexpression, and their potential as therapeutic vulnerabilities.

To test if ecDNA spatial clustering enhances transcription, they designed a plasmid with a PVT1 promoter-driven NanoLuc reporter (PVT1p-nLuc) and a TK promoter-driven Firefly luciferase control (Fig. 1b). In ecDNA+ COLO320-DM cells, the activity of PVT1p was 25-fold higher than that from minimal promoters and 4-fold higher than in ecDNA− COLO320-HSR cells (Fig. 1c). Suppression of PVT1p activity by low dose JQ1 (which breaks up ecDNA hubs) was strong in COLO320-DM (5-fold) but mild in COLO320-HSR (2-fold) (Fig. 1c). DNA/RNA FISH analysis revealed that PVT1p-driven NanoLuc transcription was enhanced in the vicinity of ecDNA hubs (Fig. 1d–f). The addition of a cis-enhancer significantly increased both reporters but ablated the differential JQ1 sensitivity between the two cell lines. These data confirm trans-activation of PVT1p by ecDNA hubs.

Tir-induced Cell Death is Dependent on Caspase-4 and GSDMD

Enteropathogenic E. coli (EPEC) is a diarrhoeagenic pathogen that colonizes intestinal cells through its type III secretion system (T3SS). One of EPEC’s effectors Tir has been shown to induce actin polymerisation and pyroptosis in macrophages but its activity in intestinal epithelial cells (IECs) is unknown. To address this, Zhong’s team utilized IECs infected with EPEC to test various parameters to confirm Tir-dependent pyroptosis including calcium chelation, caspase-4/GSDMD knockdown, and translocation of NleF, a caspase-4 inhibitor.

Results showed that caspase inhibitors (zVAD/YVAD) reduced EPEC-1-induced IEC death, confirming pyroptosis, while apoptosis (PARP1 cleavage) and necroptosis (Nec1/NSA) were ruled out (Fig 2D, S2). Caspase-4 silencing or NleF expression blocked cell death, and GSDMD cleavage was detected (Fig 2E–I). GSDMD knockdown significantly suppressed pyroptosis (Fig 2K–M), though residual death (~15%) remained unexplained. To confirm a causal relationship between GSDMD and cell death, three other IEC lines with relatively lower abundance of GSDMD (1.9–36.7×, less than SNU-C5, Fig 2N, left) but similar levels of caspase-4 were assessed (HT-29, SNU-C2B, COLO-320-HSR). All three had undetectable levels of caspase-5. Moreover, all IEC lines had intact EPEC adhesion and Tir translocation into cells. IFNγ-primed SNU-C5 and HT-29 had elevated EPEC-1-induced death relative to EPEC-0, while COLO-320-HSR and SNU-C2B with low levels of GSDMD were resistant to Tir-dependent death (Fig 2N, right). The abundance of GSDMD directly correlated with Tir-induced pyroptosis.