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G9a Epigenetically Suppresses CXCL10 Expression and Inhibits Anti-tumor Immunity in Hepatocellular Carcinoma

ABSTRACT: The immunosuppressive tumor microenvironment, characterized by limited immune cell infiltration, represents a major challenge for effective immunotherapy in hepatocellular carcinoma (HCC). Epigenetic dysregulation has emerged as a critical mechanism underlying tumorigenesis and progression; however, the specific epigenetic mechanisms governing immune infiltration remain poorly understood. Here, we investigated the role of G9a, a histone methyltransferase catalyzing H3K9 methylation, in modulating anti-tumor immunity in HCC. Bioinformatic analysis of human HCC datasets revealed a significant inverse correlation between G9a expression and T cell infiltration. Genetic ablation of G9a in hepatoma cells markedly enhanced CD8+ T cell recruitment and activation in immunocompetent mouse models. Through RNA sequencing and functional validation, we identified CXCL10 as a key chemokine directly repressed by G9a. Mechanistically, G9a mediates H3K9 dimethylation at the CXCL10 promoter, and G9a deletion or inhibition significantly reduced this repressive mark, resulting in increased CXCL10 expression and secretion. Importantly, neutralization of CXCL10 abolished G9a inhibition-induced enhancement of CD8+ T cell migration. In preclinical models, pharmacological inhibition of G9a with UNC0642 not only suppressed tumor growth by promoting T cell infiltration but also synergized with anti-PD1 therapy to achieve superior therapeutic efficacy. These findings establish G9a as an epigenetic regulator of anti-tumor immunity in HCC and provide evidence for combining G9a inhibitors with immune checkpoint blockade to improve outcomes for HCC patients who receiving immunotherapy.

ORGANISM(S): Mus musculus

PROVIDER: GSE298822 | GEO | 2025/10/31

REPOSITORIES: GEO

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Project description:Hypoxia is one of the major driving forces mediating tumor angiogenesis, aggressiveness, as well as resistance to chemo- and radiotherapy. It has also been suggested to play important roles in stem cell maintenance for both normal and cancer tissues. However, the mechanisms by which hypoxia-driven epigenetic changes modulate tumorigenesis remain poorly understood. As the histone H3 lysine 9 (H3K9) demethylase Jmjd1a and methyltransferase G9a are upregulated downstream targets of hypoxia, we focused on these two catalytically opposing epigenetic modifiers to address this question. Through the use of homozygous Jmjd1a and G9a knockout mouse embryonic stem (ES) cells, we found that Jmjd1a was not required for stem cell self-renewal and that anti-angiogenesis related genes were epigenetically dysregulated in both Jmjd1a- and G9a deficient ES cells under hypoxic conditions, accompanied by corresponding changes in H3K9 dimethylation and H3K4 trimethylation levels in the proximal promoter regions of these target genes. Most importantly, these genetic alterations led to opposing tumor phenotypes: loss of Jmjd1a results in increased tumor growth, whereas loss of G9a produces smaller tumors. These findings provide new insights on the importance of hypoxia signalling in regulating the epigenetic status and expression of angiogenesis genes that promote tumor progression. 63 microarray samples consisting of 7 mouse ES cell lines of which 2 are wild type (control), 2 Jmjd1a knockout, 2 G9a knockout and 1 G9a knockout that was reconstituted for G9a (G9a control). Each cell line and condition was seeded at 3 different densities (2X10^5, 4X10^5 and 6X10^5) in 6 cm dishes to control for the effects of cell confluency on gene expression. 18 hours after plating, the cells were subjected to normoxia (21% O2) for 24 hours (control), normoxia 20 hours followed by hypoxia (1% O2) for 4 hours (acute hypoxia) and 24 hours hypoxia (chronic treatment). Total RNA was harvested from all samples for microarrays after the 24 hour treatments.

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Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.

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G9a Epigenetically Suppresses CXCL10 Expression and Inhibits Anti-tumor Immunity in Hepatocellular Carcinoma

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