The Protein Phosphatase Inhibitor LB100 Targets the Mesenchymal Lineage of Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains a therapeutic challenge, and the aggressive basal‐like/mesenchymal subtype is particularly refractory to chemotherapy, underscoring the need for novel therapies. Leveraging genetic screens, we identified protein phosphatase 2A (PP2A) catalytic subunit PPP2CA as a target. Pharmacological PP2A inhibition selectively impaired the growth of mesenchymal PDAC cells. To delineate the mechanisms underlying sensitivity to the PP2A inhibitor LB100, we employed a dual‐pronged strategy. Functional characterization revealed metabolic reprogramming coupled with endoplasmic reticulum (ER) stress and cell death induction. Genome‐wide genetic screens identified key modifiers of LB100 sensitivity, implicating transcriptional regulators, mRNA processing, translation, and metabolism. Based on expression data linking PP2A to splicing and transcriptional regulation, we prioritized these processes for validation. Mesenchymal PDAC cells exhibited enhanced splicing following PP2A inhibition. Notably, we identified enhanced transcriptional elongation upon LB100 treatment, particularly of short genes, driven by cyclin‐dependent kinase 9 (CDK9). Our findings support a reciprocal regulatory relationship between PP2A and CDK9 that connects to the activation of ER stress response factors, including activating transcription factor 4 (ATF4). These results establish PP2A as a druggable target in mesenchymal PDAC cells and reveal a role of LB100‐induced transcriptional elongation and splicing, providing a mechanistic basis to guide future therapy development.