Genome Engineering to Prospectively Investigate the Pathogenesis of MLL-AF9 Acute Leukemia

Chromosomal rearrangements involving the MLL gene occur in both primary and treatment-related leukemias, and are associated with a poor prognosis. While animal models of MLL-AF9 translocations have improved our understanding of the role of MLL oncogenes in leukemia pathogenesis, in this study a more representative approach based on generation of endogenous activated oncogenes is used to more faithfully model the genomic events on human leukemia. This system allows for prospective investigation of the downstream effects of the initiating event on gene expression, epigenetic regulation, stem cell biology, and genome stability in order to understand the key steps critical for the pathogenesis of MLL-rearranged leukemias. We designed a set of TALENs that cut in intron 11 of the MLL gene (MLL-11 TALENs) to facilitate insertion of sequences encoding for AF9 and the fluorescent marker gene coding NeonGreen (knock-in approach). This resulted in MLL-AF9 expression controlled through the endogenous MLL promoter. Co-expression of MLL-11 TALENs with the AF9 knock-in template resulted in AF9 NeonGreen insertion both in K562 cells and in primary human hematopoietic stem cells isolated from umbilical cord blood. This approach showed a knock-in efficiency of ~20% (range 2-40%) in K562 and CD34+ cells based on NeonGreen expression detected by flow cytometry and confocal microscopy. Long-term culture of primary human hematopoietic cells showed increased frequency of knock-in cells suggesting a survival advantage. Remarkably, further enrichment of knock-in cells was promoted by colony-forming assays in semi-solid medium. Subsequent transplantation into NSG mice was performed to assess their leukemogenic potential. Strikingly, leukemia was induced within 8-14 weeks post transplantation. All mice presented with a similar disease profile that included peripheral blood blasts and splenomegaly. Histologic examination confirmed extensive replacement of bone marrow cells and splenic infiltration by leukemic blasts expressing MLL-AF9 detected by RT-PCR. The leukemic blasts showed increased levels of common MLL target genes (HoxA9, Meis1) compared to non-MLL leukemias and comparable levels to known MLL-AF9 cell lines (Mono Mac 6, THP-1). The blast cells displayed a pre-lymphoid phenotype with CD10+/CD19++/CD38++/CD34+ and were negative for mature B cell markers CD20 and IgM as measured by flow cytometry. Our studies establish a novel experimental model to generate MLL leukemia deriving from primary human hematopoietic stem cells expressing the fusion oncogene under control of the endogenous promoter. The model will allow for further prospective study of leukemia initiating and stem cell biology for a genetic subtype of poor prognosis leukemia.