Non-enzymatic Rnaseh2c orchestrates proliferating macrophage-driven immunosuppression and HCC progression via Cdk9 proliferation axis and CCL2/CCR2-mediated CD8+ Tex infiltration: a novel therapeutic paradigm with "Rnaseh2c-In1" inhibitor.

Tumor-associated macrophages are pivotal drivers of hepatocellular carcinoma (HCC) progression. However, the functional contributions of proliferating macrophages (Prolif Ms) within the tumor microenvironment (TME) remain poorly defined. Here, we integrated single-cell RNA sequencing, Cleavage Under Targets and Tagmentation (CUT&Tag), bulk RNA sequencing, multiparametric immunofluorescence, flow cytometry, and molecular dynamics simulation to dissect the phenotype, functional landscape, and targeting strategy of Prolif Ms in HCC. We identified a novel Prolif Ms subset characterized by high expression of proliferation markers (Cdk1, Mki67), distinct from classical M1/M2 macrophages. Mechanistically, ribonuclease H2 subunit C (Rnaseh2c) acts as a non-enzymatic regulator, binding directly to the cyclin-dependent kinase promoter to drive macrophage proliferation while suppressing endocytic and antigen-presenting capabilities. Rnaseh2c further promotes CD8+ exhausted T cell (CD8+ Tex) infiltration via the C-C motif chemokine ligand 2/C-C motif chemokine receptor type 2 (CCL2/CCR2) axis, fostering an immunosuppressive TME. Notably, Rnaseh2c exhibits cancer-promoting effects in both macrophages and hepatocytes. We developed a specific inhibitor "Rnaseh2c-In1", which inhibits HCC growth by reducing CCL2 secretion and CD8+ Tex infiltration, demonstrating favorable pharmacokinetics and low toxicity. This study elucidates a non-enzymatic Rnaseh2c-driven mechanism regulating Prolif Ms, providing a novel therapeutic target and candidate compound for HCC treatment.