In Vivo Profiling of Leukemic Stem Cell Fitness

By transplanting in competition multiple pre-leukemic clones in a cohort of primary recipients, we observed emergence of dominant clones during the development of AML. By sampling “winner” clones at different time points, we could observe the gradual acquisition a of cell-intrinsic growth advantage that was preserved in secondary transplantation, whereas control “loser” clones grew at a significantly lower rate. Functional characterization of the winner clones demonstrated a higher engraftment rate in vivo, whereas the self-renewal potential and growth rate in vitro was similar among winner and loser clones. By comparing the gene expression profile of leukemic stem cells isolated from various clones, we observed that the most aggressive clones share a common signature characterized by activation of multiple metabolic pathways, such as steroid biosynthesis and response to oxidative stress. Overall design: A pre-leukemic cell line was generated by isolating murine granulocyte-macrophage progenitors from a male B6.SJL-Ptprc Pepc/BoyJ and infecting with a MSCV-MLL/AF9-IRES-NeoR retrovirus. Single clones of that cell line were labeled with lentiviruses driving the expression of multiple fluorescent proteins (iRFP, GFP, Sapphire and BFP). This pool of clones was transplanted in multiple recipients (M784, M798 and M800). Disease competition and evolution was tracked prospectively in primary recipients through repeated sampling of blood and bone marrow at different time points (T0: pre-transplantation; T2: 65 days; T3: 88 days; T4:98 days). Secondary leukemic cell lines were established from individual clones, harvested at serial time points during growth in vivo. In order to study the cell-intrinsic characteristics acquired following exposure to the bone marrow microenvironment, these secondary cell lines were transplanted into additional recipients to measure engraftment potential, growth rate, cell cycle and apoptosis, gene expression profile and acquisition of secondary mutations. Leukemic burden was defined as fraction of leukemic cells in BM of secondary recipients.