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

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 or MLL-AF9 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. Transcriptomic profiling revealed breakpoint- and partner-specific gene expression patterns that influence the characteristics of the disease. 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. Our model enables mechanistic studies across MLL fusion variants and may guide the development of targeted therapies for MLL-rearranged leukemias.