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 KMT2A ( MLL ) rearrangements are aggressive hematologic malignancies associated with a poor prognosis, especially in infants. The majority of MLL breakpoints are located within the breakpoint cluster region spanning exons 8-14, with AFF1 ( AF4 ) and MLLT3 ( AF9 ) being the most frequent fusion partners. To study the contribution of different fusion partners and breakpoint locations to leukemogenesis, we created a human CRISPR/Cas9-based model. We introduced MLL::AF4 or MLL::AF9 fusions with MLL breakpoints in intron 9 or 11, respectively, into human cord blood-derived CD34 + cells from the same donor. Compared to healthy control cells, all MLL -rearranged cells showed increased proliferation and stemness, as well as an altered immunophenotype characterized by the upregulation of leukemic markers. Transcriptomic profiling revealed breakpoint- and partner-specific gene expression patterns that influence the characteristics of the disease. Notably, even after prolonged in vitro culture MLL(intron 9)::AF9 cells displayed robust colony formation in semisolid media and engrafted robustly in NOD scid gamma mice. The cells still exhibited high lineage plasticity, switching from a myeloid to a B-lymphoid identity in vivo . In conclusion, this model enables the mechanistic dissection of MLL fusion variants in vitro and in vivo , providing a foundation for developing targeted therapies for MLL -rearranged leukemias.