PD-L1 Intracellular RNA binding function regulates immune suppression

PROJECT DESCRIPTION/ABSTRACT Programmed death ligand 1 (PD-L1), which promotes immune escape, is overexpressed in triple negative breast cancer (TNBC), an aggressive subtype of breast cancer characterized by poor prognosis. Clinically approved PD-L1 antibodies augment anti-tumor immunity by blocking extracellular PD-1/PD-L1 binding. However, the contribution of intracellular PD-L1 to anti-tumor immunity and therapeutic resistance has remained poorly understood. We have discovered a novel role for intracellular PD-L1 as an RNA binding protein that promotes the stability of target RNAs. This new intracellular PD-L1 function in regulating RNA expression was independent of the established extracellular role of PD-L1 as the ligand for PD-1. The activity of the anti-tumor immune response is governed by a balance between immune effector cells and immune suppressor cells. Regulatory T cells (Tregs) are a CD4+ T cell subpopulation that inhibit effector cell activity, suppress anti-tumor immunity and promote therapeutic resistance. A hallmark of Tregs is the expression of the transcription factor, Foxp3, which binds to the promoters of genes that support Treg activity. Foxp1 is a closely related family member of Foxp3. Emerging data has demonstrated that Foxp1 cooperates with Foxp3 to encourage Foxp3-mediated transcription and Treg function by maintaining Foxp3 occupancy at promoters of target genes. In our preliminary data, we have identified Foxp1 as a key PD-L1 target RNA. We have found that the PD-L1 cytoplasmic domain, but not the PD-L1 extracellular domain, interacts with Foxp1 RNA and promotes Foxp1 expression. In addition, we have discovered that intracellular PD-L1’s promotion of Foxp1 expression is necessary for proper Treg differentiation, Treg function, and TNBC progression. The Akt-mammalian target of rapamycin (mTOR) pathway regulates metabolic reprogramming for proper T cell maturation and function. Our preliminary data suggests that PD-L1 and Foxp1 are required for proper Akt-mTOR pathway activation and metabolism specifically in Tregs, but not effector T cells, suggesting that targeting the PD-L1-Foxp1 pathway may preferentially inhibit Tregs to address therapeutic resistance. Our overarching hypothesis is that intracellular PD-L1 stabilizes Foxp1 RNA to promote Treg immunosuppressive activity and therapeutic resistance. Further, inhibiting intracellular PD-L1 will promote anti-TNBC immunity by blocking Treg activity. This hypothesis will be tested in a series of three aims: Aim 1 will determine the influence of intracellular PD-L1 on Foxp1 mRNA stability and Treg differentiation; Aim 2 will determine the effect of intracellular PD-L1 on Treg function; Aim 3 will compare intracellular PD-L1 and extracellular PD-L1/PD-1 directed TNBC therapy. Clear delineation of the impact of intracellular PD-L1 on cancer therapy will provide important insight for optimizing combination strategies aimed at overcoming immune escape and therapeutic resistance, laying the foundation for the design of the next generation of TNBC clinical trials.