Understanding PD-L1/L2 protein regulation, detection and signaling to predict melanoma therapeutic sensitivity

项目摘要/摘要 肿瘤中抗PD-1反应性的调节器和效应器包括T细胞渗透，一种免疫- 抑制性微环境、肿瘤突变负荷和肿瘤细胞内在途径(如- 连环蛋白)改变。细胞表面PD-L1水平已被认为是一种基线预测标志 抗PD-1反应性和细胞表面PD-L2水平可能具有独立于PD-1的预测价值 L1。在黑色素瘤中，MAPK抑制剂治疗强烈诱导肿瘤中PD-L1/L2的表达水平， 基质细胞和免疫细胞，提示对适应性抵抗有贡献。细胞表面的诱导 PD-L1是获得性免疫抵抗的中心，是获得性抗PD-1的机制之一 抵抗。此外，表面PD-L1蛋白稳定性的调节也与肿瘤有关 免疫监测，其中降解增加增强肿瘤特异性T细胞活性。 我们假设，更好地理解控制细胞表面PD的调控机制- L1/L2的稳定性、临床检测和肿瘤细胞固有的促生存信号可以揭示 黑色素瘤免疫逃避和治疗反应。我们将使用蛋白质组学方法 解剖这些过程并探索黑色素瘤表面糖蛋白组PD-L1/L2 相互作用体和细胞质信号小体，以提名机制和/或标记 治疗反应性。我们将反复询问临床肿瘤样本和同基因小鼠 BRAF、NRAS和NF1突变型黑色素瘤模型。黑色素瘤的这些免疫活性模型是 考虑到其紫外线诱导的高突变负荷，临床上对CD8 T细胞的依赖 对广泛转移的治疗反应和能力，包括脑转移。 我们将使用含唾液酸聚糖的细胞表面标记来分析活细胞表面 糖蛋白质组，PD-L1/L2免疫共沉淀富集相互作用，以及基于APEX的 用蛋白质组学方法原位确定细胞内动态PD-L1/L2邻域相互作用 对PD-1结扎或干扰素治疗的反应。我们将讨论调节PD-L1/L2泛素化的因素， 循环和降解，糖基化如何影响膜PD-L1免疫组织化学 检测去糖基化是否改善基线和On-1的抗PD-1反应的预测 新的PD-L1/L2胞浆基序如何介导PD-1依赖的肿瘤细胞 生存信号。通过对临床黑色素瘤的邻近结扎试验，我们将检测PD-L1/L2 肿瘤细胞固有的信号转导降低了治疗效果。这些蛋白质组学方法应该会取得进展 我们对转移性黑色素瘤治疗反应模式的理解和预测 生物标志物和组合靶标。

PROJECT SUMMARY/ABSTRACT Modulators and effectors of anti-PD-1 responsiveness in cancer include T cell infiltration, an immune- suppressive microenvironment, tumor mutational burden, and tumor cell-intrinsic pathway (e.g., - catenin) alterations. Cell-surface PD-L1 level has been implicated as a baseline predictive marker of anti-PD-1 responsiveness, and cell-surface PD-L2 level may have predictive value independent of PD- L1. In melanoma, MAPK inhibitor therapy strongly induces PD-L1/L2 expression levels in tumor, stromal and immune cells, suggesting contributions to adaptive resistance. Induction of cell-surface PD-L1 is central to adaptive immune resistance and implicated as a mechanism of acquired anti-PD-1 resistance. Moreover, the regulation of surface PD-L1 protein stability has been implicated in tumor immune surveillance, where increased degradation augments tumor-specific T cell activity. We hypothesize that a better understanding of regulatory mechanisms controlling cell-surface PD- L1/L2 stability, clinical detection, and tumor cell-intrinsic pro-survival signaling could shed insights into melanoma immune evasion and therapeutic responsiveness. We will use proteomic approaches to dissect these processes and to explore the melanoma surface glycoproteome, the PD-L1/L2 interactome and the cytoplasmic signalosome in order to nominate mechanisms and/or markers of therapeutic responsiveness. We will interrogate iteratively clinical tumor samples and syngeneic mouse models of Braf, Nras and Nf1 mutant melanoma. These immune-competent models of melanoma are clinically relevant given their UV-induced high mutational burdens, dependence on CD8 T cells for therapeutic responses and capability for widespread metastases, including metastases to the brain. We will use cell-surface labeling of sialic acid-containing glycans to analyze the live cell surface glycoproteome, co-immunoprecipitation of PD-L1/L2 to enrich for interactomes, and APEX-based proteomic strategy to define in situ dynamic intracellular PD-L1/L2 neighborhood interactomes in response to PD-1 ligation or IFN treatment. We will address what regulate PD-L1/L2 ubiquitination, recycling and degradation, how glycosylation affects membrane PD-L1 immunohistochemical detection, whether deglycosylation improves prediction of anti-PD-1 responses at baseline and on- treatment, and how novel cytoplasmic motifs of PD-L1/L2 mediate PD-1-dependent tumor cell pro- survival signaling. Using proximity ligation assays on clinical melanoma, we will test whether PD-L1/L2 tumor cell-intrinsic signaling reduces therapy efficacy. These proteomic approaches should advance our understanding of therapy response patterns in metastatic melanoma and nominate predictive biomarkers and combinatorial targets.