Mechanistic understanding of Wiskott-Aldrich syndrome protein (WASp)-mediated epigenetic regulation of T helper cell differentiation in acute leukemia.

Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells, yet its role in leukemia remains poorly defined. This study investigates the epigenetic regulation of key immune genes by T helper (T_H) cell transcription factors. Human CD4⁺ T cells from healthy donors and patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) were transfected with control CRISPR (CO), WASp knockout (WKO), and WASp overexpression (WOE) plasmids. Flow cytometry confirmed intracellular WASp staining in CO, WKO, and WOE CD4⁺ T cells. WOE leads to elevated mRNA levels of T_H1-associated transcription factors, such as TBX21 and IFNγ. Notably, TBX21 enrichment increased significantly in WOE cells, while no such enrichment was observed for GATA3, RORC, or FOXP3. In addition, aberrant accumulation of R-loops at key gene loci, including TBX21, STAT1, ATM, and H2AFX, was prominently observed in WKO cells but not in WOE conditions. DNA methylation increased WKO cells but m6A levels were elevated under WOE conditions. Immunofluorescence analysis revealed much higher WASp expression in WOE but increased γH2A.X fluorescence in WKO. We found WASp stably interacts with the STAT1 binding region in the TBX21 promoter by molecular docking studies. Nitric oxide, reactive oxygen species, and glutathione levels were higher in WKO cells, while lactate dehydrogenase levels were increased in WOE samples. These findings collectively imply that WOE in acute leukemia promotes T_H1 cell activity in a significant manner through epigenetic regulation and could be a potential therapeutic target.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04474-4.