PD-L1 Intracellular RNA binding function regulates immune suppression

项目描述/摘要 促进免疫逃逸的程序性死亡配体1（PD-L1）在三阴性乳腺癌中过表达 乳腺癌（TNBC）是乳腺癌的一种侵袭性亚型，其特征在于预后差。临床批准 PD-L1抗体通过阻断细胞外PD-1/PD-L1结合来增强抗肿瘤免疫力。但 细胞内PD-L1对抗肿瘤免疫和治疗抗性的贡献仍然很差 明白我们已经发现细胞内PD-L1作为RNA结合蛋白的新作用， 靶RNA的稳定性。这种新的细胞内PD-L1调节RNA表达的功能是独立的， PD-L1作为PD-1配体的既定细胞外作用。抗肿瘤免疫活性 免疫应答由免疫效应细胞和免疫抑制细胞之间的平衡控制。调节性T T细胞（T细胞）是抑制效应细胞活性、抑制抗肿瘤免疫并 促进治疗抵抗力。转录因子Foxp 3的表达是TGFAP的一个标志， 与支持Treg活性的基因的启动子结合。Foxp 1是Foxp 3的一个密切相关的家族成员。 新出现的数据表明，Foxp 1与Foxp 3合作，促进Foxp 3介导的转录 Treg通过维持Foxp 3在靶基因启动子处的占据而发挥功能。在我们的初步数据中， Foxp 1是PD-L1的关键靶RNA。我们已经发现PD-L1胞质结构域，而不是 PD-L1胞外结构域与Foxp 1 RNA相互作用并促进Foxp 1表达。另外我们有 发现细胞内PD-L1对Foxp 1表达的促进作用是Treg正常分化所必需的， Treg功能和TNBC进展。Akt-哺乳动物雷帕霉素靶蛋白（mTOR）通路调节 代谢重编程以促进适当的T细胞成熟和功能。我们的初步数据表明，PD-L1 Foxp 1和Foxp 1是Tcells中适当的Akt-mTOR通路激活和代谢所必需的，但不是 效应T细胞，这表明靶向PD-L1-Foxp 1通路可能优先抑制TcR，以解决 治疗抵抗我们的总体假设是，细胞内PD-L1稳定Foxp 1 RNA， 促进Treg免疫抑制活性和治疗抗性。此外，抑制细胞内 PD-L1将通过阻断Treg活性促进抗TNBC免疫。这一假设将在一系列测试中得到验证 目的1：确定细胞内PD-L1对Foxp 1 mRNA稳定性和Treg的影响。 目标2将确定细胞内PD-L1对Treg功能的影响;目标3将比较 细胞内PD-L1和细胞外PD-L1/PD-1导向的TNBC疗法。明确界定的影响 细胞内PD-L1在癌症治疗中的作用将为优化联合策略提供重要见解， 在克服免疫逃逸和治疗抗性方面，为下一个 TNBC的临床试验。

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.