Combined pan and precision targeting in extrachromosomal DNA-positive gastric cancer

Gastric adenocarcinoma (GAC) remains a major health challenge with high mortality due to late diagnosis, therapy resistance, and limited targeted options. Extrachromosomal DNA (ecDNA)-circular DNA elements carrying amplified oncogenes such as MYC, ERBB2, and EGFR-enhances transcriptional output, therapy resistance, and genomic instability. However, its role in GAC is poorly understood, and current models fail to recapitulate patient tumor complexity. Patient-derived organoids (PDOs) preserve tumor-specific features, including ecDNA, providing an ideal platform for mechanistic and therapeutic exploration.We hypothesize that ecDNA drives unique oncogenic programs in GAC that create exploitable vulnerabilities. Analysis of 221 TCGA-STAD samples with AmpliconArchitect identified ecDNA in 33.3% of primary tumors, absent in matched normal blood. ecDNA+ tumors showed distinct amplification patterns, with proteomic profiling revealing CHEK1 and TFRC enrichment, suggesting co-targetable vulnerabilities. To model these tumors, we established a PDO biobank comprising 5 primary and 9 ascites-derived PDOs. ecDNA was detected in 5/9 (55.6%) ascites-derived and 1/5 (20%) primary PDOs, consistent with ecDNA enrichment in metastatic settings. Amplified oncogenes included PTP4A3, for which inhibitors are available.RNA-seq comparison of ecDNA+ PDO 385 with ecDNA- PDOs (4666, 4601) revealed distinct transcriptional programs, with enriched expression on chromosomes 8 and 20. Transcription factor analysis identified MYC, also amplified as ecDNA. Immunofluorescence confirmed higher TFRC, CHEK1, MYC, and PRL3 (PTP4A3) expression and increased Ki67 in ecDNA+ PDO 385, supporting oncogene overexpression and aggressive growth. Drug screening predicted compounds reversing the ecDNA+ signature. Functional assays showed the TFRC inhibitor (TfR-1-IN-1) reduced proliferation of ecDNA+ PDO 385, while CHK1 inhibition had modest effects. Combined TFRC and CHK1 inhibition synergistically suppressed ecDNA+ PDO growth, with minimal effects on ecDNA- controls. These results highlight the selective vulnerability of ecDNA-driven tumors to co-targeting strategies. Using a PDO biobank, we aim to uncover and exploit vulnerabilities in ecDNA-positive GAC.