Chrysosplenetin-induced TMED3 aggregation triggers unfolded protein response in pancreatic cancer

Abstract

Pancreatic cancer (PC) continues to demand urgent therapeutic innovation given its limited treatment options. Here, through phenotypic screening of a natural product library followed by systematic validation, we identified chrysosplenetin (CHR) as a bioactive compound with anti-PC activity. Transcriptomic profiling and functional analyses demonstrated that CHR induced endoplasmic reticulum (ER) stress, thereby activating the unfolded protein response (UPR) and subsequent apoptosis, while paradoxically triggering a protective autophagy. Genetic or pharmacological inhibition of autophagy potentiated CHR-induced antitumor efficacy. Using an integrated approach including proteomic analysis, bio-layer interferometry, cellular thermal shift assay, and molecular docking, we confirmed TMED3 as a direct target of CHR. Functional studies revealed that disruption of TMED3 expression partially restored ER homeostasis, attenuating CHR-induced UPR activation and apoptosis. Furthermore, CHR combined with standard chemotherapy or autophagy inhibitors exhibited enhanced antitumor activity in preclinical models, providing a basis for future therapeutic exploration of the TMED3–ER stress axis. Together, our findings establish TMED3 as a novel therapeutic target in PC, revealing that disrupting ER proteostasis via TMED3 perturbation represents a potential therapeutic strategy warranting further investigation. Chrysosplenetin (CHR) induces TMED3 aggregation, causing lethal ER stress in pancreatic cancer and identifying TMED3 as a direct target and druggable vulnerability. CHR combined with gemcitabine or autophagy inhibitors enhances antitumor activity in preclinical models. Phenotypic screening identifies CHR as a bioactive compound with anti-pancreatic cancer activity. CHR induces ER stress-mediated apoptosis but also triggers protective autophagy, which limits its antitumor activity. TMED3 is validated as a direct target of CHR, linking its disruption to UPR activation and apoptosis. Combining CHR with gemcitabine or autophagy inhibitors enhances antitumor activity in PDX and PDO models. Phenotypic screening identifies CHR as a bioactive compound with anti-pancreatic cancer activity. CHR induces ER stress-mediated apoptosis but also triggers protective autophagy, which limits its antitumor activity. TMED3 is validated as a direct target of CHR, linking its disruption to UPR activation and apoptosis. Combining CHR with gemcitabine or autophagy inhibitors enhances antitumor activity in PDX and PDO models. Chrysosplenetin (CHR) induces TMED3 aggregation, causing lethal ER stress in pancreatic cancer and identifying TMED3 as a direct target and druggable vulnerability. CHR combined with gemcitabine or autophagy inhibitors enhances antitumor activity in preclinical models.