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 TCF3/TCF3::HLF DNA occupancy profiles of parental HAL-01 and wild-type TCF3 KO HAL-01. Chromatin prepared from parental HAL-01 cells (TCF3_IP_Parental) and TCF3-KO HAL-01 cells (TCF3_IP_Knockout) was prepared for ChIP assay using E2A (CST #12258) antibody for pull-down. ChIP-seq outputs were processed to generate bigwig files using bamCoverage from deepTools and call peaks using MACS2 to visualize the enrichment landscapes with the deepTools software.