CASM-DEPENDENT FLCN SEQUESTRATION ENHANCES METABOLIC FLEXIBILITY IN PANCREATIC CANCER

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the simultaneous upregulation of biosynthetic pathways and catabolic programs, including lysosomal biogenesis and autophagy driven by the MiT/TFE transcription factors. In normal cells, the master regulatory kinase mTORC1 phosphorylates and inhibits MiT/TFE factors under pro-anabolic conditions. However, in PDAC cells, MiT/TFE factors largely evade mTORC1-dependent phosphorylation, accumulate in the nucleus, and activate downstream catabolic, pro-tumorigenic pathways. The molecular elements responsible for the uncoupling of MiT/TFE factors from mTORC1 are unknown. Here, we show that dysregulation of the Folliculin (FLCN):FLCN-interacting protein (FNIP) complex, an obligate factor required for mTORC1-mediated MiT/TFE phosphorylation, leads to constitutive activation of catabolic programs in PDA. Unlike the total loss of FLCN observed in Birt-Hogg-Dubé tumor syndrome, FLCN and FNIP1/2 expression is not reduced in PDAC cells and patient samples. Instead, FLCN:FNIP accumulates at the lysosome, where FLCN is unable to exert its GAP activity on its client protein, the RagC GTPase, thereby preventing mTORC1-dependent phosphorylation of MiT/TFE factors. Supporting this model, forced expression of a GDP-locked RagC mutant (S75N) bypasses the requirement for FLCN and restores mTORC1-mediated TFEB regulation. Mechanistically, we found that FLCN sequestration at lysosomes depends on conjugation of Atg8 proteins to single membranes (a process known as CASM), and that blocking this conjugation successfully restores cytoplasmic localization of both FLCN-FNIP and TFEB. Critically, CASM-dependent FLCN sequestration is driven, at least in part, through the pathological accumulation of cholesterol within lysosomes in PDAC cells, a condition typically associated to the Niemann-Pick type C (NPC) disease. Reducing lysosomal cholesterol was sufficient to rescue FLCN localization and normalize TFEB regulation. Altogether, these findings identify lysosomal cholesterol accumulation as a key mechanism by which MiT/TFE factors evade mTORC1 surveillance, promoting the coupled anabolic-catabolic programs that support PDAC progression.