TCF3::HLF orchestrates an enhancer-promoter network with activation of MEF2C to promote an immature HSC gene expression program in leukemia.
Oncogenic fusion transcription factors (TFs) frequently drive hematopoietic malignancies by altering gene expression in key developmental programs. TCF3::HLF is a fusion TF that characterizes a rare, treatment-resistant subtype of B-cell acute lymphoblastic leukemia (t(17;19) TCF3::HLF-positive B-ALL). Despite its clinical significance, the mechanisms by which TCF3::HLF induces leukemia are unclear. We used HiChIP mapping and genetic interference to analyze TCF3::HLF at the 3D-genome level, revealing enhancer-promoter interactions that control gene activation or repression. Notably, TCF3::HLF directly regulates MEF2C expression through its enhancer, as interference disrupted MEF2C transcription and inhibited leukemia propagation. This disruption also diminished embryonal hematopoietic stem cell (HSC) gene signatures and restored mature HSC and B-lymphoid markers. These findings highlight MEF2C as a critical component of the transcriptional network reprogrammed by TCF3::HLF. Our study provides insight into how TCF3::HLF rewires the 3D genome to drive leukemia and serves as a resource for further exploration of the TCF3::HLF regulome. Overall design: To delineate the 3D-genomic interactions orchestrated by the fusion protein TCF3::HLF in t(17;19) ALL cells, we knocked out the  TCF3 wild-type allele, thereby enabling selective immunoprecipitation of the fusion protein when using antibodies against TCF3.  We compare the RNAseq expression profile for wild-type TCF3 knockout  HAL-01 with parental HAL-01 control samples to verify that no major changes occure. We included 697 with shSCR as an additional control representing the TCF3::PBX1 fusion protein. Expression datasets for parental HAL-01 cells, TCF3-KO HAL-01 cells (TCF3_KO) and 697 cells with shSCR (X697) were used to generate PCA plots visualizing the variability among the cell lines.