Abstract
BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) exhibits significant heterogeneity, with therapy resistance in Activated B-cell-like (ABC) and relapsed Germinal Center B-cell-like (GCB) subtypes being major challenges, with underlying drivers poorly understood. METHODS: We performed single-cell RNA sequencing (scRNA-seq) on de novo ABC-DLBCL, de novo GCB-DLBCL, and relapsed GCB-DLBCL specimens. Analyses included malignant cell subclustering, R-loop scoring, trajectory inference, and cell-cell communication. HMGN2 was functionally validated in vitro. RESULTS: We identified an aggressive, poor-prognosis B-cell subpopulation in de novo ABC and relapsed GCB-DLBCL, defined by significantly reduced R-loop formation.This phenotype was directly associated with the downregulation of High-Mobility Group Nucleosome Binding Domain 2 (HMGN2). Mechanistically, we validated that HMGN2 promotes R-loop formation; its loss derepresses IL4R expression, leading to hyperactivation of the oncogenic PI3K-AKT signaling pathway. Functional validation confirmed that HMGN2 knockdown in DLBCL cell lines enhanced proliferation and clonogenicity, whereas its overexpression was inhibitory. The tumor microenvironment in relapsed GCB tumors exhibited profoundly immunosuppressive features, including functionally impaired CD8(+)T cells and dominant inhibitory BTLA-TNFRSF14 interactions. CONCLUSIONS: Our study identifies a potential HMGN2/R-loop/PI3K-AKT axis that may drive malignant cell-intrinsic fitness while shaping an immune-suppressive microenvironment. These findings position HMGN2 as a candidate regulator of DLBCL progression and a potential prognostic biomarker and therapeutic target to address treatment resistance. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-026-08064-7.