Elucidating and overcoming therapeutic resistance in esophageal adenocarcinoma

Background: Esophageal adenocarcinoma (EAC) accounts for over 80% of esophageal cancers in the United States and remains a highly lethal malignancy with poor prognosis and rising incidence. Despite advances in therapy, recurrence occurs in most EAC patients, largely due to the emergence of therapeutic resistance. Resistance can be intrinsic or acquired from a rare subpopulation known as Drug Tolerant Persister (DTP) cells, which survive cytotoxic therapy through non-genetic adaptive mechanisms. Unlike genetic resistance, the drug-tolerant state is reversible upon drug withdrawal. This study aims to establish cisplatin-derived DTP(CDDP-DTP) and drug-resistant (CDDP-DR) EAC cell models to elucidate resistance mechanisms, identify therapeutic vulnerabilities, and develop strategies to overcome resistance and prevent recurrence in EAC. Methods: CDDP-DTP cells were generated by exposing EAC cells to the IC₈₀ of CDDP for four days, while CDDP-DR cells were developed via stepwise dose escalation based on the IC₅₀. Cytotoxic effects of compounds targeting ferroptosis, pyroptosis, and epigenetic regulation (RSL3, DMB, BIX-01294, and GSK3326595) were evaluated by MTS assays in four EAC cells (FLO-1, OE19, OE33, SK-GT-4). Drug synergy was analyzed using the Combenefit tool. BH3 profiling was performed to assess mitochondrial apoptotic priming and anti-apoptotic dependency. Seahorse assay was used to evaluate cellular metabolism. Cell cycle distribution and cell death were analyzed by propidium iodide (PI) and annexin V/PI staining. Gene and protein expression were measured by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. Results: CDDP-DTP EAC cells exhibited reduced proliferation and upregulation of canonical DTP markers, while CDDP-DR cells displayed >2-fold increased resistance to CDDP, elevated ATF4 expression, an epithelial-to-mesenchymal transition (EMT) phenotype, and resistance to apoptosis accompanied by metabolic reprogramming. Among compounds targeting non-apoptotic, non-genetic adaptive mechanisms, RSL3, DMB and BIX-01294 effectively suppressed EAC cell growth compared with GSK3326595. Notably, BIX-01294 combined with CDDP and DMB combined with a Bcl-xL inhibitor exhibited synergistic cytotoxicity in EAC cells. The target proteins of these compounds-GPX4, GSDMD, and G9A-were upregulated in both TCGA datasets and EAC cells, further highlighting their potential roles as mediators of therapeutic resistance and as promising therapeutic targets. Conclusions: This study successfully established CDDP-DTP and CDDP-DR models of EAC and revealed that targeting non-apoptotic, adaptive mechanisms can enhance therapeutic response. These findings lay the foundation for multi-omic characterization of resistant EAC populations and support exploration of non-apoptotic vulnerabilities to undermine recurrence and improve long-term outcomes in EAC.