Silibinin is a natural molecular glue that inhibits PD-L1 glycomaturation

BACKGROUND: The adaptive upregulation of the immune-checkpoint PD-L1 in response to interferon-γ (IFNγ) protects solid tumors from cytotoxic lymphocytes and undermines adoptive cell immunotherapies. Currently, pharmacologically interrupting the trafficking of nascent PD-L1 to the surface of cancer cells is not feasible in a clinical setting. HYPOTHESIS/PURPOSE: We investigated whether silibinin (SBN), the primary bioactive flavonolignan found in Silybum marianum (milk thistle) seeds, could impede the post-translational glycomaturation of PD-L1 and enhance T-cell-mediated antitumor cytotoxicity. METHODS: We assessed the effects of SBN on PD-L1 glycomaturation by integrating electrophoretic mobility shifts, Endo-H/PNGase-F glycosidase sensitivity mapping, transcript-level glyco-enzyme arrays, computational molecular dynamics (MD), biochemical cross-linking, AlphaLISA binding, and flow cytometry. Co-cultures of cancer cells with cytokine-activated T cells were performed using SBN as a single agent or in combination with the small-molecule PD-L1 inhibitor BMS-1166. RESULTS: SBN converted nascent PD-L1 into an Endo H-sensitive, 43 kDa, high-mannose species that becomes trapped in the endoplasmic reticulum (ER), regardless of STAT3 status. SBN did not alter CD274/PD-L1 transcription or global N-glycosylation. MD predicted, and BS3 cross-linking confirmed, weak but significant SBN-induced PD-L1 dimerization which diminished PD-1 binding. SBN increased the ability of BMS-1166, which also induces PD-L1 dimerization and ER retention of underglycosylated PD-L1, to block PD-1/PD-L1 interaction and to eradicate IFNγ-elicited plasma-membrane PD-L1. SBN was synthetically lethal with BMS-1166, causing multiple cancer types to become highly responsive to the immunocytolytic activity of T cells. CONCLUSION: SBN is a plant-derived molecular glue that intercepts PD-L1 glycomaturation co-translationally in the ER. When combined with canonical PD-L1 dimerizers, SBN collapses adaptive PD-L1 expression and renders cancer cells exquisitely susceptible to the cytolytic insults of T cells. Due to its favorable safety profile and oral bioavailability, SBN is a promising "glycotherapeutic" phytochemical for T-cell-based immunotherapy, particularly in IFN-rich tumors with reactive PD-L1 expression programs.