Role of MNX1-mediated histone modifications and PBX gene family in MNX1-induced leukemogenesis.

Aberrant MNX1 upregulation is a hallmark of pediatric t(7;12)(q36;p13) AML. In this study, we investigated the downstream targets of MNX1 and their relationship to MNX1-induced histone modifications. Using a comprehensive multi-omics approach combining Tandem Mass Tag–based (TMT) mass spectrometry, RNA-Sequencing, qPCR, antibody-guided chromatin tagmentation sequencing (ACT-Seq), assay for transposase-accessible chromatin using sequencing (ATAC-Seq), and ChIP–qPCR, we identified Pbx1 as a direct MNX1 target and Pbx4 and Pbxip1 as secondary targets associated with leukemic progression. MNX1 binds directly to the Pbx1 promoter, increasing H3K4me3 and reducing H3K27me3 marks, consistent with promoter activation. Despite the transient nature of MNX1 promoter binding, these histone modifications persisted, suggesting a “hit-and-run” epigenetic remodeling mechanism. During leukemic progression, a global increase in H3K4me3 was observed, consistent with broad epigenetic activation. Genome-wide motif analysis further revealed that MNX1 motifs were most enriched within H3K4me1-marked enhancer regions, while ATAC and H3K4me3 enrichment correlated with open chromatin containing PBX family motifs, particularly Pbx4 motifs. These findings suggest that PBX transcription factors might have stage-specific roles in MNX1-driven leukemogenesis, with Pbx1 functioning as an early, MNX1-dependent effector, and Pbx4 and Pbxip1 contributing at later stages through secondary mechanisms. Treatment with the methyltransferase inhibitor Sinefungin, previously shown to block MNX1-mediated histone methylation and leukemogenesis, significantly reduced MNX1-induced Pbx1 expression, consistent with promoter-specific, methylation-dependent regulation at this early preleukemic stage, whereas Pbx4 and Pbxip1 remained unaffected, supporting their roles as secondary targets during leukemic progression. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-36367-8.