SQSTM1/p62-mediated PD-L1 biomolecular condensate formation promotes lung tumorigenesis.

[INTRODUCTION] Biomolecular condensates are membraneless organelles functionally involved in diverse processes, including cancer progression. Sequestosome-1 (SQSTM1)/p62 regulates liquid-liquid phase separation to drive biomolecular condensates, while programmed death-ligand 1 (PD-L1) promotes cancer cell growth. Although SQSTM1 has been shown to form condensates with several complexes, its full range of partners remains incompletely characterized.

[OBJECTIVES] This study aimed to investigate the role of SQSTM1 in mediating PD-L1 condensate formation and its contribution to lung tumorigenesis.

[METHODS] The SQSTM1-PD-L1 interaction and condensate colocalization were examined by immunoprecipitation and immunofluorescence. Truncated SQSTM1 constructs validate the interacting domain with PD-L1, which is supported by computational AlphaFold3 analysis. We applied 1,6-hexanediol, FDA-approved PD-L1 antibody Atezolizumab, and CRISPR/Cas9-based SQSTM1 knockout to disrupt SQSTM1/PD-L1 biomolecular condensates, along with in vitro and in vivo experiments. The Survivin levels were analyzed using real-time quantitative PCR and immunoblotting. Clinical correlations of SQSTM1 and PD-L1 expression with prognosis were assessed using public datasets.

[RESULTS] Our research demonstrate that SQSTM1, by its Phox1 and Bem1 (PB1) domain, directly interacts with PD-L1 to facilitate the SQSTM1/PD-L1 biomolecular condensate formation. The formation of SQSTM1/PD-L1 condensates prevents PD-L1 from ubiquitin-proteasome-mediated degradation and stabilizes PD-L1 levels. Deletion of the PB1 domain in SQSTM1 inhibits the formation of PD-L1 biomolecular condensates and induces PD-L1 K48 ubiquitination for protein degradation. Disruption of SQSTM1/PD-L1 condensates with 1,6-hexanediol induces PD-L1 proteolysis and reduces cell viability. Knockout of SQSTM1 disrupts PD-L1 condensate formation, downregulates PD-L1 protein levels, and attenuates tumor growth. Treatment with PD-L1 antibody drug Atezolizumab inhibits SQSTM1/PD-L1 condensates and suppresses tumor formation. Clinically, high SQSTM1 and PD-L1 expression correlated with poor prognosis in lung cancer patients.

[CONCLUSION] Together, the SQSTM1 directly interacts with non-membrane-associated PD-L1 to drive the formation of SQSTM1/PD-L1 biomolecular condensates, which prevent PD-L1 degradation, promote lung tumorigenesis, and serve as a potential therapeutic target in lung cancers.