Parkin Induces Ubiquitination and Large Extracellular Vesicle Release of HMGB1 to Activate Antitumor Immunity

Abstract Parkin is a mitochondria-associated E3 ubiquitin (Ub) ligase that mediates mitophagy and organelle quality control. More recently, Parkin has been implicated in stimulating antitumor immunity and reprogramming the tumor immune microenvironment. Here, we showed that Parkin ubiquitinates the alarmin molecule, high mobility group box-1 (HMGB1) on Lys146 (K146) using predominantly K48 linkages. By molecular modeling, the in-between-ring (IBR) domain of Parkin (Gln326-Leu358) made extensive contacts with the amino-terminus A box of HMGB1 (Met1-Ser42), forming a mitochondria-associated Parkin-HMGB1 complex that juxtaposes K146 to Ub active site residues Gly76 and Arg74. Instead of proteasomal degradation, Parkin ubiquitination of K146 enabled the loading of HMGB1, but not HMGB1 K146A mutant, onto autophagy- and mitochondria-derived large extracellular vesicles (LEV). In turn, released Parkin-HMGB1 LEV stimulated a potent interferon (IFN) and cytokine response in recipient cells, expanding CD8+ T cell subsets with effector (CD69+/KLRG1+), self-renewal (TCF-1+/PD-1+), and cytotoxic (KLRG1+/GrzB+) properties. Conditional expression of Parkin induced HMGB1 release, activated intratumoral CD8+ T cells, and suppressed syngeneic tumor growth in vivo in a response that was abolished by HMGB1 silencing. These data identify that Parkin-LEV regulated release of HMGB1 reprograms antitumor immunity via stimulation of IFN signaling and expansion of specialized CD8+ T cell subsets.