PTK2/FAK inhibition triggers TMED9-mediated protective autophagy in pancreatic cancer cell via enhancing ERGIC-ERES contact

Macroautophagy/Autophagy represents a promising therapeutic target in oncology, exhibiting context-dependent roles in tumor progression. Kinase inhibitors are a large group of anti-cancer drugs, and elucidating the regulatory effects of different kinase inhibitors on Autophagy offers a valuable strategy to advance our understanding of Autophagy regulation in Cancer and develop innovative anti-cancer therapies. In this study, we developed a drug screening platform and performed high-content screening using a kinase inhibitor library. The screening identified PTK2/FAK (protein tyrosine kinase 2) inhibitors as potent Autophagy inducers. Mechanistic investigations revealed that PTK2/FAK inhibition triggers rapid autophagic flux through a non-classical mechanism dependent on PTK2/FAK expression, which is associated with the spatial redistribution of ER exit sites (ERES). Further analysis demonstrated that PTK2/FAK Inhibitor (FAKi)-induced ERES-associated Autophagy is not contingent upon full PREB/SEC12 and SEC16 expression, but is sensitized to TMED9 expression level. Additionally, we identified AKAP13 as a novel FAKi-responsive protein that undergoes dephosphorylation upon FAKi treatment and contributes to TMED9-mediated ERES-associated Autophagy. Given that ERES-associated Autophagy is initiated through membrane contact between ERES and the ER-Golgi intermediate compartment (ERGIC), we further observed enhanced TMED9-ERGIC interactions following FAKi treatment. Furthermore, functional studies confirmed that FAKi-induced Autophagy promotes pancreatic ductal adenocarcinoma (PDAC) cell survival both in vitro and in vivo. Collectively, our work unveils a previously unrecognized mechanism of FAKi-mediated Autophagy induction and provides new insights for developing targeted therapies against PDAC.Abbreviations: AKAP13: A-kinase anchoring protein 13; AMPK: AMP-activated protein kinase; BAF: bafilomycin A1; ECM: extracellular matrix; ER: endoplasmic reticulum; ERES: endoplasmic reticulum exit sites; ERGIC: ER-Golgi intermediate compartment; GSK: GSK2256098; MTOR: mechanistic target of rapamycin kinase; PDAC: pancreatic ductal adenocarcinoma; PI3K: phosphoinositide 3-kinase; PI4KB: phosphatidylinositol 4-kinase beta; PTK2/FAK: protein tyrosine kinase 2; TMED9: transmembrane p24 trafficking protein 9; ULK1: unc-51 like Autophagy activating kinase 1.