ZFX Drives T-cell Leukemia Growth Through m5C Methylation of PD1 mRNA

Zinc finger protein X-linked (ZFX) and programmed cell death protein 1 (PD1) are significantly upregulated in T-cell acute lymphoblastic leukemia (T-ALL), according to a study published in Biological Procedures Online on 26 October 2026. The work links the two molecules through an RNA modification pathway: ZFX activates the m5C methyltransferase NSUN2, which stabilizes PD1 mRNA by m5C methylation, thereby promoting the aggressiveness of this highly aggressive hematological malignancy that currently has limited treatment options.

The researchers analyzed clinical specimens alongside two T-ALL cell lines, Jurkat and MOLT4, using immunohistochemistry, reverse transcription quantitative polymerase chain reaction (RT-qPCR), and western blotting to assess ZFX and PD1 expression and global m5C levels. Both ZFX and PD1 were elevated in T-ALL samples and cell lines, and this upregulation correlated with elevated global m5C levels, pointing to a role for RNA methylation in the disease.

To probe function, the team knocked down ZFX using sh-ZFX and measured the consequences with cell counting kit-8 (CCK-8) proliferation assays, apoptosis assays, Transwell migration assays, and reactive oxygen species (ROS) measurements. ZFX knockdown suppressed cell proliferation and migration while increasing apoptosis and ROS, and it also reduced m5C levels, indicating that ZFX supports malignant cell behavior partly through maintaining RNA methylation.

Mechanistic experiments combined chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) with luciferase reporter assays. These showed that ZFX binds to the NSUN2 promoter at the region spanning positions -1719 to -1706 and transcriptionally activates NSUN2 expression. NSUN2, an m5C methyltransferase, in turn promotes m5C-dependent stabilization of PD1 mRNA, completing a regulatory chain from a transcription factor to an RNA modification enzyme to a stabilized immune-checkpoint transcript.

Rescue experiments tested the chain directly: co-transfecting sh-ZFX cells with NSUN2 overexpression plasmids reversed the effects of ZFX knockdown, restoring PD1 expression, m5C modification, and tumor aggressiveness in vitro. In vivo validation used a syngeneic C57BL/6 mouse T-ALL tumor model, in which NSUN2 overexpression likewise restored tumor aggressiveness after ZFX knockdown. The authors conclude that ZFX promotes T-ALL progression by regulating NSUN2-mediated m5C modification of PD1, and they suggest ZFX and NSUN2 as potential therapeutic targets for T-ALL. As the article is shared early as a peer-reviewed, accepted version of record pending further edits, the findings should be read in that context.

Subject of Research: The role of ZFX-regulated NSUN2-mediated m5C methylation of PD1 mRNA in T-cell acute lymphoblastic leukemia progression.

Article Title: ZFX Promotes the Progression of T-cell Acute Lymphoblastic Leukemia by Regulating NSUN2-Mediated m5C Methylation of PD1 mRNA

Article References: Song, C.-M., Jian, F.-H., Wang, X., Wei, J., Liu, G.-H., Wang, S.-B., Liu, J., Zhang, B., & Wang, C.-Y. (2026). ZFX Promotes the Progression of T-cell Acute Lymphoblastic Leukemia by Regulating NSUN2-Mediated m5C Methylation of PD1 mRNA. Biological Procedures Online. https://doi.org/10.1186/s12575-026-00372-y

Image Credits: AI Generated

DOI: 10.1186/s12575-026-00372-y

Keywords: T-cell acute lymphoblastic leukemia, ZFX, NSUN2, m5C methylation, PD1, RNA modification, mRNA stabilization, ChIP-qPCR, Jurkat, MOLT4, C57BL/6 mouse model, therapeutic targets