Immune escape signatures of Ming infiltrative gastric cancer (GC) driven by super-enhancers (SEs). (A) Hematoxylin-eosin staining of GC. (B) SE peaks of H3K27ac histone modifications. (C) Distribution of H3K27ac SE peaks. (D) Venn diagrams of SE-driven genes and chromosomal landscape of infiltrative SE-driven genes. (E) Gene Ontology-Kyoto Encyclopedia of Genes and Genomes (GO-KEGG) pathway enrichment for SE-driven genes. (F) Unsupervised hierarchical clustering using 16 prognostic genes. (G) Kaplan–Meier survival curves of two subgroups. (H) Immune infiltration analysis. (I) Immunohistochemical (IHC) scores of programmed death-ligand 1 (PD-L1) in 70 GC tissues. TSS, Transcription Start Site; TTS, Transcription Termination Site.

Expression levels of ZFP36L1 in infiltrative GC driven by ZFP36L1-SE. (A) Friends analysis of 16 SE-driven prognostic genes. (B) Correlations between clinical characteristics and the ZFP36L1 mRNA expression in TCGA. (C) Tumor Immune Dysfunction and Exclusion scores in high and low expression levels of ZFP36L1 groups. (D) Correlation between immune infiltration cells and the mRNA expression level of ZFP36L1 in TCGA. (E) Correlation between ZFP36L1 mRNA expression and immune checkpoints in TCGA. (F) H3K27ac signals of SEs and target genes in infiltrative GC. (G) H3K27ac signals of ZFP36L1-SE in GC. (H) Protein expression of ZFP36L1 in 12 tumor and paired adjacent normal tissues of patients with infiltrative GC. (I) Protein expression of ZFP36L1 in six GC cell lines. (J) Expression level of ZFP36L1 after SE inhibition treatment. (K) H3K27ac signals of ZFP36L1-SE after SE inhibition treatment.

ZFP36L1 promotes IFN-γ-induced PD-L1 expression. (A) mRNA and (C) protein expression of PD-L1 in GC cell lines with or without ZFP36L1 knockdown. (B) mRNA and (D) protein expression of PD-L1 in GC cell lines with or without ZFP36L1 overexpression. (E) Fluorescent signal of the PD-L1 membrane protein in GC cell lines with or without ZFP36L1 knockdown and (F) overexpression.

SPI1 binds to the ZFP36L1-SE region and drives the regulation of PD-L1. (A) Schematic of transcription factor motif enrichment in ZFP36L1-E1. (B) Kaplan–Meier survival plot of SPI1 in TCGA. (C) mRNA and (D) protein expression of ZFP36L1 after transcription factors (TFs) plasmid transfection. (E) Correlation between SPI1 and PD-L1 in TCGA. (F) PD-L1 protein expression in simultaneous SPI1 overexpression and ZFP36L1 knockdown cells. (G) Prediction of SPI1–BRD4– P300 binding on the STRING website. Co-immunoprecipitation between (H) exogenous SPI1 and BRD4 in 293T cells , or (I) endogenous SPI1 and BRD4 in MGC803. (J) SPI1 directly interacts with BRD4 in vitro by GST pull-down experiment. (K) ZFP36L1-E1 binding of different TFs detected using dual-luciferase assay. (L) SPI1 enriched regions in ZFP36L1-E1 detected using dual-luciferase assay. (M) Different binding sites of SPI1 in ZFP36L1-E1 detected using dual-luciferase assay. (N) Wild-type and motif-deletion mutant E1C binding of SPI1 detected using dual-luciferase.

ZFP36L1 positively regulates PD-L1 by activating HDAC3 mRNA decay. (A) Word cloud of predicted ZFP36L1 target genes. (B) The mRNA expression of predicted target genes in MGC803 cell overexpressing ZFP36L1. (C) HDAC3 protein expression in ZFP36L1 knockdown cells. (D) Effect of HDAC3 on CD274 promoter activity in 293T using dual-luciferase assay. (E) Correlation between changes of histone H3K27 acetylation and PD-L1 protein expression in MKN45 cells overexpressing HDAC3. (F) The histone H3K27 acetylation levels of CD274 promoter regions using ChIP assay. (G) Correlation between changes of histone H3K27 acetylation and ZFP36L1 protein expression. (H) The H3K27ac and SPI1 enrichment of ZFP36L1-E1C regions using ChIP assay. (I-J) PD-L1 protein expression in simultaneous ZFP36L1 and HDAC3 overexpression cells. (M) HDAC3 mRNA decay in ZFP36L1 knockdown and overexpression cells after actinomycin D treatment. (N) ZFP36L1 mRNA-binding level by RNA-binding protein immunoprecipitation. (O) ZFP36L1 mRNA-binding site in AU-rich element (ARE) of 3ʹUTR confirmed using dual-luciferase assay. (P) CCCH-type zinc finger domain of ZFP36L1 protein binding to HDAC3 mRNA confirmed using RNA pull-down assay. ARE, adenylate uridylate-(AU-) rich element; 3ʹUTR, 3ʹ untranslated region.

Positive correlation between ZFP36L1 and PD-L1 in vivo. (A-C) IHC staining of SPI1, ZFP36L1 and HDAC3 protein in 70 PD-L1-positive GC tumor tissues. (D) Schematic diagram of experiments in C57BL/6J and BALB/c-nu mice. (E) Subcutaneous tumor weight from mice injected with control and ZFP36L1 knockdown cells. (F) The PD-L1 mRNA expression in subcutaneous tumors. (G) IHC staining of ZFP36L1 and PD-L1 protein in subcutaneous tumors. (H) Number of pulmonary metastases in different groups of C57BL/6J mice after tail vein injection of control and ZFP36L1 knockdown cells. (I) Hematoxylin & eosin (HE) and IHC staining of CD8α in pulmonary metastases. (J) Number of pulmonary metastases in different groups of BALB/c-nu mice after tail vein injection of control and ZFP36L1 knockdown cells.

Schematic diagram of SPI1–ZFP36L1–HDAC3–PD-L1 signaling axis (created with gdp.renlab.cn).