GluProRS Inhibition Abolishes Cancer Stem Cells in Pancreatic Cancer by Inducing the Integrated Stress Response and Countering TGFβ/Activin-SMAD2/3-HIF1A

The developmentally dynamic stem cell-like subpopulation in cancers known as cancer stem cells (CSCs) metastasize efficiently, form new tumours, and are resistant to current therapeutics. Understanding the molecular mechanisms of the stem cell-like state could help eliminate CSCs via novel therapeutic applications in aggressive cancers such as pancreatic ductal adenocarcinoma (PDAC). Here, we performed a compound screen uncovering that inhibition of Hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) supports CSCs, whereas a novel proline-uncompetitive glutamyl-prolyl-tRNA synthetase (GluProRS) inhibitor NCP26 eliminates CSCs. By using a multiomic strategy with transcriptomics and quantitative secretomic analyses, we uncovered that TGFβ/Activin signalling induces HIF1A-mediated pseudohypoxia via ɑ-Ketoglutarate depletion due to P4HA1 intracellular upregulation. In turn, HIF1A and SMAD2/3 form a transcriptional complex that regulates CSC self-renewal, invasiveness and chemoresistance. NCP26 has several anti-tumour effects, including direct activation of the Integrated Stress Response (ISR) and indirect inhibition of the pseudo-hypoxia circuitry mediated by SMAD2/3-HIF1A that deposits collagen in ECM. Furthermore, GluProRS is secreted from PDAC cells and elevated in PDAC patient blood compared to healthy and pancreatitis patients. Collectively, the secreted GluProRS protein could be a useful biomarker for early PDAC detection, and its inhibitor NCP26 is an attractive candidate for combined chemotherapeutic applications.