Establishing Human Niche Xenograft Models for Myeloid and Lymphoid Leukemia Driven By MLL-AF9

The MLL-AF9 fusion oncogene is found in pediatric leukemia of both the myeloid and lymphoid lineage, while in adults the same oncogene is predominantly associated with myeloid leukemia. We have been able to establish in vitro models that faithfully recapitulate these patient phenotypes by lentiviral transduction of MLL-AF9 into CD34⁺ cells from fetal Cord Blood and adult Bone Marrow. Upon transplantation of transduced MLL-AF9 expressing cells into NOD-SCID/NSG mice, serially transplantable lymphoid leukemia could be generated within 15-24 weeks, but the myeloid engraftment was more difficult to achieve and CD33⁺/CD19^- myeloid clones were only observed in about 10% of the cases. Importantly, these CD33⁺/CD19^- clones displayed impaired self-renewal as indicated by the lack of secondary transplantation capacity while lymphoid CD33^-/CD19⁺ clones readily engrafted in secondary recipients to give rise to 2^nd ALL. These results are in contrast with what we detect in vitro or in patients where both myeloid and lymphoid phenotypes are observed. We hypothesized that extrinsic and species-specific factors might dictate the lineage fate of the leukemic clone, and therefore we have started to use a new model in which human mesenchymal stem cells (MSCs) are coated on scaffolds, which are then implanted subcutaneously into NSG mice to allow vascularization and formation of a “human niche” (huScaffold). Within these human niches we observe both AML and ALL upon transplantation of MLL-AF9-transduced CB CD34⁺ cells. Moreover, the human niche also allowed engraftment and tumor formation of primary AML MLL-AF9 patient samples. These data indicate that extrinsic factors present in the human niche can dictate lineage fate of the leukemia. Future directions include further improvements of the human niche model like using MSCs that we have genetically engineered to express factors like IL3 and TPO.

In addition, we took advantage of the CB MLL-AF9 in vitro and in vivo models to test the efficacy of a small molecule inhibitor of the BET family named I-BET151. I-BET151 targets BRD3/4 association to acetylated histones and thereby prevents MLL-AF9 binding to chromatin. We demonstrate that I-BET151 inhibits the growth of CB MLL-AF9 cells in an in vitro MS5 co-culture setting, under both myeloid and lymphoid conditions. Furthermore, we transplanted CD19⁺ MLL-AF9 clones into secondary recipients and we performed intra-scaffold injections of the inhibitor as soon as the tumors were palpable. Tumor volume was measured during the treatment and we observed a significant 2.5-fold reduction of tumor volume in a dose-dependent manner.

In conclusion, our data indicate that extrinsic factors can dictate lineage fate of MLL-AF9-driven leukemia. The humanized in vivo xenograft model allows the faithful recapitulation of human disease and thus will aid in the development of novel therapeutic approaches to treat leukemias driven by oncogenes such as MLL-AF9. Ongoing studies include those in which we target the FLT3 pathway since our transcriptome studies indicated that this tyrosine kinase receptor is highly upregulated in MLL-AF9-positive leukemias. In vitro targeting of this receptor abrogated both initiation as well as maintenance of transformation by MLL-AF9. We analyzed FLT3L-initiated genome-wide transcriptional changes in MLL-AF9 cells and observed that inhibition of the FLT3 pathway resulted in reduced expression of genes such Bcl2 and CyclinD2, possibly involving loss of activity of the PI3K pathway, thereby inducing apoptosis in MLL-AF9 cells.