Leukemia is the most common cancer in children, with the first genetic alterations often occurring during fetal development. These initiating events generate preleukemic cells, which are the evolutionary ancestors of leukemia that arises after birth. Because of our inability to directly access human fetal preleukemia, the identity of the cell of origin and the steps of leukemia evolution remain largely unknown. Down syndrome leukemogenesis represents a disease setting to study human preleukemia and the evolutionary steps that lead to fully transformed leukemia. Up to 30% of children with Down syndrome (trisomy 21) exhibit a preleukemic transient abnormal myelopoiesis (TAM) and, overall, have a 150-fold increased risk of developing myeloid leukemia within the first 5 years of life. However, the mechanism by which an extra copy of chromosome 21 predisposes to preleukemia and leukemia remains unclear.

Understanding Down syndrome leukemogenesis requires a humanized model that faithfully recapitulates the full developmental spectrum of premalignant and malignant stages of Down syndrome leukemia. Using CRISPR/Cas9–mediated gene editing in human disomic and trisomic fetal liver–derived hematopoietic stem and progenitor cells and xenotransplantation, we developed a model with which to characterize the genetic events and cellular contexts underlying the preleukemic and leukemic phases of Down syndrome leukemogenesis.

Trisomy 21 hematopoietic stem and progenitor cells (HSPCs) showed reduced proliferation in vitro and generated smaller grafts in xenotransplanted mice, with reduced serial transplant ability, as compared with that of disomic HSPCs. Preleukemia was initiated in trisomy 21, but not disomic, long-term hematopoietic stem cells (LT-HSCs) when mutations in the erythroid-megakaryocyte transcription factor GATA binding protein 1 (GATA1) were introduced, which led to exclusive expression of the short isoform (GATA1s). Subsequent leukemic progression could occur in multiple stem and progenitor populations, was independent of trisomy 21, and induced through deletion of cohesin genes, including STAG2 (STAG2ko). Serial engraftment in mice showed that GATA1s-induced preleukemia underwent spontaneous resolution, which contrasted with the persistent ability of the GATA1s/STAG2ko–induced leukemia to engraft serially in mice. Leukemic progression was developmentally restricted to fetal and early postnatal stages; adult-derived bone marrow HSPCs were unable to undergo GATA1s/STAG2ko-induced leukemic transformation. We identified a molecular mechanism by which three chromosome 21 microRNAs (miRNAs) contributed to the predisposition toward preleukemia initiation. Simultaneous overexpression of miR-99a, miR-125b-2, and miR-155 in normal disomic LT-HSCs recapitulated a trisomy 21–like hematopoietic state, as assessed through comparable lineage differentiation, reduced self-renewal capacity, and similar gene expression and open chromatin accessibility profile. Removal of these miRNAs in trisomy 21 LT-HSCs inhibited GATA1s-induced preleukemia development. Using secondary xenotransplantations of defined cell populations, we identified CD117⁺/KIT proto-oncogene (KIT) as a marker of disease-driving cells. Pharmacological KIT inhibition targeted preleukemic stem cells, both in GATA1s-induced preleukemia and in primary Down syndrome preleukemia patient samples.

Collectively, our results provide insight into how human preleukemia and leukemia evolve in fetal life and early childhood. We were able to identify …