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

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy with limited treatment options. Zinc finger protein X-linked (ZFX) and programmed cell death protein 1 (PD1) have been implicated in tumor progression, but their roles in T-ALL and their regulatory interplay with RNA methylation (m5C) remain unclear. Clinical specimens and T-ALL cell lines (Jurkat, MOLT4) were analyzed using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-qPCR), and western blot to assess ZFX/PD1 expression and m5C levels. Functional consequences of ZFX knockdown (sh-ZFX) were investigated through cell counting kit-8(CCK-8) proliferation, apoptosis assay, Transwell migration, and reactive oxygen species (ROS) assays. Mechanistic studies integrated Chromatin Immunoprecipitation (ChIP)-qPCR assay and luciferase reporter assay. Rescue experiments were performed by co-transfecting sh-ZFX with NSUN2 overexpression plasmids. In vivo validation utilized a syngeneic C57BL/6 Mouse T-ALL tumor model. ZFX and PD1 were significantly upregulated in T-ALL samples and cell lines, correlating with elevated global m5C levels. ZFX knockdown suppressed cell proliferation, migration, and m5C levels while increasing apoptosis and ROS. Mechanistically, ZFX bound to the NSUN2 (m5C methyltransferase) promoter (-1719~-1706) and transcriptionally activated NSUN2 expression, further promoting the m5C-dependent stabilization of PD1 mRNA. NSUN2 overexpression reversed ZFX knockdown effects, restoring PD1 expression, m5C modification, and tumor aggressiveness in vitro and in vivo. Our study demonstrated that ZFX promoted the progression of T-ALL by regulating NSUN2-mediated m5C modification of PD1, suggesting ZFX and NSUN2 as potential therapeutic targets for T-ALL.T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematological malignancy with limited treatment options. Zinc finger protein X-linked (ZFX) and programmed cell death protein 1 (PD1) have been implicated in tumor progression, but their roles in T-ALL and their regulatory interplay with RNA methylation (m5C) remain unclear. Clinical specimens and T-ALL cell lines (Jurkat, MOLT4) were analyzed using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-qPCR), and western blot to assess ZFX/PD1 expression and m5C levels. Functional consequences of ZFX knockdown (sh-ZFX) were investigated through cell counting kit-8(CCK-8) proliferation, apoptosis assay, Transwell migration, and reactive oxygen species (ROS) assays. Mechanistic studies integrated Chromatin Immunoprecipitation (ChIP)-qPCR assay and luciferase reporter assay. Rescue experiments were performed by co-transfecting sh-ZFX with NSUN2 overexpression plasmids. In vivo validation utilized a syngeneic C57BL/6 Mouse T-ALL tumor model. ZFX and PD1 were significantly upregulated in T-ALL samples and cell lines, correlating with elevated global m5C levels. ZFX knockdown suppressed cell proliferation, migration, and m5C levels while increasing apoptosis and ROS. Mechanistically, ZFX bound to the NSUN2 (m5C methyltransferase) promoter (-1719~-1706) and transcriptionally activated NSUN2 expression, further promoting the m5C-dependent stabilization of PD1 mRNA. NSUN2 overexpression reversed ZFX knockdown effects, restoring PD1 expression, m5C modification, and tumor aggressiveness in vitro and in vivo. Our study demonstrated that ZFX promoted the progression of T-ALL by regulating NSUN2-mediated m5C modification of PD1, suggesting ZFX and NSUN2 as potential therapeutic targets for T-ALL.