<i>Supporting data for “</i><b>FASN inhibition complements PD-L1 checkpoint blockade by suppressing palmitoylation of MHC-I in hepatocellular carcinoma</b><i>”.</i>

The advent of immune checkpoint inhibitors (ICIs) has revolutionized the treatment approach for hepatocellular carcinoma (HCC). However, patients with lowered major histocompatibility complex class I (MHC-I) expression continue to exhibit resistance to ICIs, and there are limited targets available for enhancing MHC-I levels. In this study, we discovered that inhibiting fatty acid synthase (FASN), either genetically or pharmacologically, elevated HCC cells' MHC-I levels. The cytotoxicity of CD8+ T lymphocytes that are specific to the antigens was activated and antigen presentation was strengthened by this increase in MHC-I. Mechanistically, FASN inhibition decreased MHC-I's palmitoylation, which in turn inhibited lysosome degradation of MHC-I. We identified the palmitoyltransferase DHHC3 as a direct binder of MHC-I, exerting a negative regulation on the levels of MHC-I protein. The lack of<i> Fasn</i> increased MHC-I levels in an orthotopic HCC mice model and facilitate CD8+ T lymphocytes infiltrating the tumor and eliminating cancer cells. Moreover, combining anti-PD-L1 antibody with two different FASN inhibitors, orlistat and TVB-2640, effectively inhibited the growth of tumors in vivo. Furthermore, it was shown that a higher percentage of cytotoxic CD8+ T cells was connected with decreased expression of FASN by multiplex immunohistochemistry of human HCC samples and bioinformatic analysis of TCGA data. The discovery of FASN as a negative regulator of MHC-I offers a rationale for combining FASN inhibitors with immunotherapy for the treatment of HCC.免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）的出现革新了肝细胞癌（hepatocellular carcinoma, HCC）的治疗策略。然而，主要组织相容性复合体I类（major histocompatibility complex class I, MHC-I）表达降低的患者仍对ICIs产生耐药，且目前可用于提升MHC-I水平的靶点十分有限。本研究发现，通过遗传或药理学手段抑制脂肪酸合酶（fatty acid synthase, FASN）可升高肝癌细胞的MHC-I水平。该MHC-I水平的提升能够激活抗原特异性CD8+ T淋巴细胞的细胞毒性，并增强抗原呈递功能。机制层面，FASN抑制可降低MHC-I的棕榈酰化修饰，进而抑制MHC-I的溶酶体降解。本研究鉴定出棕榈酰转移酶DHHC3（palmitoyltransferase DHHC3）为MHC-I的直接结合蛋白，可负向调控MHC-I蛋白水平。在原位肝细胞癌小鼠模型中，*Fasn*基因缺失可升高MHC-I水平，促进CD8+ T淋巴细胞浸润肿瘤并清除癌细胞。此外，将抗PD-L1抗体与两种不同的FASN抑制剂奥利司他（orlistat）和TVB-2640联合使用，可有效在体内抑制肿瘤生长。进一步通过人类肝癌样本的多重免疫组化分析及癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据的生物信息学分析发现，细胞毒性CD8+ T细胞占比更高与FASN表达降低显著相关。本研究揭示FASN作为MHC-I的负调控因子，为将FASN抑制剂与免疫治疗联合用于肝细胞癌治疗提供了理论依据。