FLT3 Signaling Enhances Stemness in Murine MLL-AF9 Acute Myeloid Leukemia.

Abstract 2980

Acute myeloid leukemia (AML) is developmentally similar to normal hematopoiesis and driven by a subpopulation of leukemia stem cells (LSCs). LSCs are defined by their ability to self-renew and differentiate into more mature leukemia cells. These properties are influenced by the stem cell niche in which they reside. A number of key cytokines within this microenvironment have been identified, including CXCL12 and stem cell factor (SCF), while others have altered the lineage fate of leukemia by in vitro manipulation of growth factor conditions. Activating mutations in fms-like tyrosine kinase receptor-3 (FLT3) have suggested a role for FLT3 signaling in LSC development and function, but little is known about the role of wild type FLT3 and its ligand (FLT3-L) in the LSC niche. In our study, we examined the impact of different hematopoietic growth factors, specifically FLT3-L, on leukemia stem cell properties in a mouse model of MLL-rearranged AML.

Murine CD117⁺ hematopoietic progenitor cells retrovirally transduced with the MLL-AF9 oncogene were selected and cultured in the presence of either FLT3-L or a combination of interleukin-3 (IL-3), interleukin-6 (IL-6), and SCF. We found that FLT3-L selectively expanded a population of CD117⁺/CD11b^lo/CD16/32^lo cells with a common myeloid progenitor (CMP)-like surface phenotype. This population was present at a frequency of only 1.3% in cells cultured in IL-3/IL-6/ SCF, with remaining cells being mostly CD117⁺/CD11b^hi/CD16/32^hi of a granulocyte-macrophage progenitor (GMP)-like surface phenotype. Wright-Giemsa staining showed that cells expanded in FLT3-L also appeared small and morphologically primitive compared to IL-3/IL-6/ SCF cells. When myeloid growth factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 were added, FLT3-L expanded cells differentiated into a heterogeneous population of CD117⁺/CD11b^hi/CD16/32^hi and CD117⁻/CD11b^hi/CD16/32^hi cells, and regained the typical myelomonocytic blast morphology seen in MLL-AF9 AML. Flow cytometry with Hoechst 33342 and Pyronin Y staining showed that the cells expanded in FLT3-L were more quiescent, with 47.1% in G0 phase compared to only 1.5% in IL-3/IL-6/SCF expanded cells. Consistent with this, FLT3-L expanded cells showed increased resistance to daunorubicin in colony forming assays. Upon transplantation into lethally irradiated mice, cells expanded in FLT3-L produced leukemia at a remarkably higher frequency (8.7 log-fold using limiting dilution analysis) than cells expanded in IL-3/IL-6/SCF. To explain the differences observed, immunoblotting revealed selective upregulation of the canonical Wnt/β-catenin pathway in FLT3-L expanded cells. In AML, Wnt signaling has been shown to mediate leukemogenesis and LSC self-renewal and homing, and thus is a plausible mechanism for regulating the leukemia stem cell properties enhanced by FLT3-L. Our findings suggest a key role for FLT3 signaling in the development and function of LSCs in MLL-AF9 AML through upregulation of the Wnt/β-catenin pathway.