程序性细胞死亡1（PD-1）和细胞性T淋巴细胞抗原4（CTLA-4）同时阻断的组合显著提高了恶性黑色素的总生存率。免疫检查点阻遏剂（ICB）限制了肿瘤的免疫逃逸，但仅适用于大约三分之一的肿瘤，在大多数情况下作用的时间也有限。目前正在研究多种克服ICB抗药性的方法，其中包括添加预期能作用于免疫和肿瘤细胞的表观遗传学药物。对乙酰基脱氧胞苷（guadecitabine）是一种脱氧核苷酸双核苷酸前体，通过磷酸二酯键与鸟苷链接，表现出良好的临床前相1试验成果，NIBIT-M4（NCT02608437）。我们采用同基因B16F10小鼠黑色素模型研究了免疫检查点阻断抗体对CTLA-4和PD-1的联合作用及其加入乙酰基脱氧胞苷的效果。利用流式细胞术，多重免疫荧光和甲基化分析，综合性地研究了不同治疗下肿瘤和宿主的反应。与ICBs相结合使用，乙酰基脱氧胞苷显著降低了皮下肿瘤的生长和转移形成，与单独ICBs和乙酰基脱氧胞苷治疗相比。特别是，在肿瘤微环境（TME）中，乙酰基脱氧胞苷通过增加效应器记忆CD8+ T细胞，在脾脏诱导效应器NK细胞，减少肿瘤浸润性调节性T细胞和骨髓源性抑制细胞（MDSC），极大地增强了联合ICBs的疗效。乙酰基脱氧胞苷与ICBs结合使用，提高了血清IFN-γ水平和具有抗血管生成活性的IFN-γ诱导趋化因子水平。乙酰基脱氧胞苷导致DNA去甲基化，特别是中间甲基化水平的位点。这些结果表明乙酰基脱氧胞苷是一种有前途的表观遗传学药物，可添加至ICBs治疗中。 ©2023. 作者们。

The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor's immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437).We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor's and the host's responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis.In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-γ and IFN-γ-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels.These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy.© 2023. The Author(s).