Identification of HSPE1 as a new actionable cancer vulnerability leads to an innovative and effective combination therapy for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest human malignancies, with limited therapeutic options and a lack of druggable vulnerabilities beyond a narrow set of oncogenic drivers. To identify cancer dependencies that are both essential in vivo and drug-tractable, we performed an unbiased genome-wide CRISPR loss-of-function screen under in vivo tumor-selective pressure. This approach revealed the mitochondrial co-chaperone HSPE1 (Hsp10) as a previously unrecognized, tumor-promoting dependency in PDAC. Genetic depletion of HSPE1 markedly impaired tumor growth, survival, and tumor-initiating capacity across multiple PDAC models in vitro and in vivo, including patient-derived xenografts. Mechanistically, HSPE1 functioned as a central survival node by engaging two parallel and targetable mitochondrial pathways. First, HSPE1 cooperated with its canonical partner HSPD1 to regulate cell-cycle progression, and apoptosis. Second, HSPE1 was functionally associated with mitochondrial dynamics, correlating with altered regulation of the OPA1/OMA1 axis, revealing an HSPD1-independent mechanism linking mitochondrial stress adaptation to cancer cell fitness. This dual signaling architecture exposes a previously unappreciated mitochondrial vulnerability selectively exploited by PDAC cells. Importantly, both HSPE1-regulated pathways were amenable to therapeutic targeting in vivo. Pharmacological inhibition of the HSPD1/HSPE1 complex or the OPA1/OMA1 pathway significantly suppressed tumor growth, while combined targeting produced robust synergistic antitumor activity in both cell line–derived and patient-derived PDAC models. Moreover, this combination strategy provides a modest yet consistent incremental benefit to standard-of-care chemotherapies, underscoring its translational relevance. Together, these findings establish HSPE1 as a bona fide cancer dependency uncovered through in vivo functional genomics, uncover a dual mitochondrial vulnerability, and provide a rational framework for combination therapy design. More broadly, this work highlights the power of in vivo CRISPR screening to directly inform therapeutic strategies and identifies mitochondrial stress adaptation as a promising and generalizable target in cancer.