The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

中文翻译：

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MDM2 配体 Nutlin-3 差异性地改变免疫阻断受体 PD-L1 和 CD276 的表达。

p53/MDM2 通路与肿瘤细胞中促癌免疫抑制受体表达之间的联系尚不清楚。在本报告中，我们评估了两种关键免疫受体 CD276 和 PD-L1 的表达是否存在 p53 和/或 MDM2 依赖性。使用邻近连接测定来量化响应 Nutlin-3 的原位蛋白质-蛋白质相互作用。使用 CRISPR-Cas9 引导 RNA 介导的基因消融创建了一组 p53 无效黑色素瘤细胞。使用流式细胞术分析评估 TP53 或 ATG5 基因消融的影响，以及 Nutlin-3 和 ATM 抑制剂对细胞表面 PD-L1 和 CD276 的影响。使用靶向 siRNA 去除 CD276，通过流式细胞术评估细胞周期参数的变化。T 细胞增殖测定用于评估 CD4+ T 细胞的活性作为 ATG5 基因型的函数。CD276 响应 Nutlin-3 与 MDM2 形成蛋白质-蛋白质相互作用，类似于已知的 MDM2 相互作用因子 p53 和 HSP70。同基因 HCT116 p53-wt/null 癌细胞证明 Nutlin-3 以 p53 依赖性方式在细胞表面诱导 CD276。PD-L1 也意外地被 Nutlin-3 诱导，但 PD-L1 不结合 MDM2。ATM 抑制剂 KU55993 在 Nutlin-3 诱导 PD-L1 的条件下降低 PD-L1 水平，表明 MDM2 和 ATM 对 PD-L1 稳态水平具有相反的作用。在 CD276 不受影响的条件下，A375 黑色素瘤细胞克隆中的 TP53 基因消除也会导致 PD-L1 上调。ATG5 基因缺失的 A549 细胞仅上调 PD-L1，进一步表明 PD-L1 和 CD276 处于不同的遗传控制之下。TP53 的基因失活或 MDM2 配体 Nutlin-3 的使用会改变免疫阻断受体 PD-L1 和 CD276 的表达。CD276 升高的生物学功能是响应 Nutlin-3 促进改变细胞周期进程，而 PD-L1 升高的主要作用是抑制 T 细胞。这些数据表明 TP53 基因状态、ATM 和 MDM2 影响细胞表面上的 PD-L1 和 CD276 旁系同源物。这些数据对于使用靶向 p53 通路的药物作为免疫检查点受体表达的修饰剂具有重要意义。