耐药是当前制约肝癌PD-1抗体疗效的关键瓶颈，相关分子机制与预警标志物亟待探索。申请人在既往发现白细胞分化抗原73（CD73）作为一种新型肝癌转移驱动分子的基础上，经预实验发现：CD73可经非酶途径上调单纯疱疹病毒侵入受体（HVEM）蛋白表达，引起肝癌PD-1抗体耐药；ERK激酶在被CD73经非酶途径激活后，可诱导HVEM T253位点发生磷酸化，抑制HVEM泛素化降解；此外，PD-1耐药患者的血清CD73、HVEM水平明显升高。本课题拟应用空间转录组测序、质谱流式细胞术等技术，从细胞、动物、临床三个层面开展研究，解析CD73激活ERK信号的具体方式；阐明CD73经非酶途径促进HVEM磷酸化修饰，进而驱动肝癌PD-1耐药的分子机制；最后，评估血清CD73、HVEM水平对PD-1抗体疗效的预测价值。预期成果可为肝癌PD-1抗体耐药提供全新的转录后修饰调控机制，并为预警耐药提供新型循环标志物。

Programmed cell death protein-1 (PD-1) is considered as one of the key therapeutic approach for hepatocellular carcinoma (HCC). Unfortunately, low responsive rate has become a severe bottleneck which greatly restricts the treatment efficacy of PD-1 antibodies, and the mechanism of drug resistance needs to be clarified urgently. In previous study, the applicant reported that CD73 was a novel driven gene of regulating HCC metastasis. Here, the applicant made some outbreaking findings from pre-experiments as follows: 1) CD73 can up regulate the expression of HVEM, a novel immune checkpoint molecule, and induce the resistant phenotype towards PD-1 antibody independent of its normal enzyme activity. 2) Meanwhile, when activated by CD73 through non enzymatic pathway, ERK kinase can induce HVEM T253 site phosphorylation and enhance its protein stability; 3) Moreover, the serum levels of sCD73 and sHVEM were significantly higher in patients who showed resistance to PD-1 treatment. On such basis, present project aims to use a variety of advanced technologies including 10x genomics spatial transcriptome sequencing, MS, and flow cytometry to carry out systematic research from the distinct levels including cell, animal and clinical, and to explore the specific molecular mechanism involved in the process of how CD73 activating ERK, how ERK enhancing the stability of HVEM protein, and thus inducing PD-1 resistance of HCC. A clinical cohort will be constructed to evaluate the predictive value of sCD73 and sHVEM for the efficacy of anti-PD-1 treatment. The expected results of present project will help to unveil a novel posttranscriptional modificationin-related regulatory mechanism for the drug resistance of anti-PD-1 treatment in HCC, and provide a new circulating tumor biomarker for alarming drug resistance.