Enhanced hemato-endothelial specification during human embryonic development through developmental cooperation between AF4-MLL and MLL-AF4 fusions

The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia (B-ALL) and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains enigmatic; MA4 is always expressed in t(4;11)+B-ALL patients, but the reciprocal fusion A4M is expressed in only 50% of patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion co-operates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors (HEPs), both endothelial-primed and hemogenic-primed. Double fusion-expressing HEPs specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-seq and H3K79me3 ChIP-seq, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing HEPs. Importantly, ChIP-seq analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion expressing differentiating hematopoietic cells. Overall these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development. RNA sequencing of EV or MA4-A4M expressing HEPS cells.