Characterizing the Impact of MLL Fusion Variants and Fusion Partners on Leukemia Plasticity Using a Human CRISPR-Engineered MLL-Rearranged Leukemia Model [RNA-Seq Classical]

Acute leukemias involving MLL (KMT2A) rearrangements are aggressive hematologic malignancies associated with poor prognosis. MLL fusions typically involve MLL exons 8-14, with AF4 (AFF1) and AF9 (MLLT3) being frequent partners. To investigate how fusion partners and breakpoint locations influence the disease, we developed a CRISPR/Cas9 model by introducing MLL-AF4 (t(4;11)) or MLL-AF9 (t(9;11)) fusions with MLL breakpoints in intron 9 or 11 into cord blood CD34+ cells. The MLL-rearranged cells showed increased proliferation and stemness, as well as an altered immunophenotype involving the upregulation of AML markers. MLL(intron 9)-AF9 cells engrafted robustly in NSG mice and showed high lineage plasticity, switching from a myeloid to a B-lymphoid identity in vivo. Transcriptomic profiling confirmed the lineage switch and the transcriptomic signature revealed progressive stages of B-cell lineage commitment in individual mice. Our model enables mechanistic studies across MLL fusion variants and may guide the development of targeted therapies for MLL-rearranged leukemias. Overall design: RNA-seq profiling of human cord blood-derived t(9;11) cells generated by CRISPR/Cas9. We compared in vitro cultured cells with ex vivo cells from the same human cord blood donors that have been transplanted into NOD scid gamma (NSG) mice and engrafted in the bone marrrow.