Background Immunotherapy represented by the programmed death-1 (PD-1)/ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. Methods In this study, we first performed a Stress and Proteostasis CRISPR interference library-based screening to identify PD-L1 positive modulators. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in malignant melanoma. Results Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. Suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We also found that PD-L1 protein abundance is regulated by YAP1/TFCP2 transcriptional complex. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 and PD-L1 transcription. Furthermore, inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent that is comparable to anti-PD-1 mAb therapies with no obvious toxicity. Conclusions These findings uncover a novel molecular mechanism for regulating PD-L1 protein abundance by a E3 ligase in cancer cells and reveal the potential of using TRAF6 inhibitors to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

中文翻译：

---

TRAF6 通过黑色素瘤中的 YAP1-TFCP2 信号传导调节 PD-L1 表达

背景以程序性死亡-1（PD-1）/配体1（PD-L1）单克隆抗体为代表的免疫治疗已引领肿瘤治疗进入新时代。然而，低总体反应率和高耐药率在很大程度上损害了现有免疫检查点疗法的临床益处。最近的研究将 PD-1/PD-L1 阻断的反应与肿瘤细胞中 PD-L1 的表达水平相关联。因此，确定控制肿瘤细胞中 PD-L1 蛋白表达和稳定性的分子靶点和途径是一个主要优先事项。方法 在这项研究中，我们首先进行了基于应力和蛋白质稳态 CRISPR 干扰库的筛选，以鉴定 PD-L1 阳性调节剂。然后，我们使用体外和体内测定来研究 TRAF6 及其下游 YAP1/TFCP2 信号在恶性黑色素瘤中的生物学功能和机制。结果 在这里，我们将 TRAF6 鉴定为黑色素瘤细胞中 PD-L1 的关键调节因子。抑制 TRAF6 表达会下调黑色素瘤细胞膜表面的 PD-L1 表达。我们还发现 PD-L1 蛋白丰度受 YAP1/TFCP2 转录复合物的调节。TRAF6 通过 K63 多泛素化修饰稳定 YAP1，随后促进 YAP1/TFCP2 和 PD-L1 转录的形成。此外，硼替佐米对 TRAF6 的抑制通过减少内源性 PD-L1 增强了 CD8+ T 细胞的溶细胞活性。值得注意的是，硼替佐米增强抗肿瘤免疫力的程度可与抗 PD-1 mAb 疗法相媲美，且无明显毒性。