Characterization of Leukemia-Initiating Cells in a Transgenic Model of Chronic Phase Chronic Myelogenous Leukemia (CML).

Abstract 858

In normal hematopoiesis, only a small population of lin-Sca-1+c-kit+ (LSK) cells with Flt3-CD150+48− immunophenotype has long-term hematopoietic stem cell (LT-HSC) capacity, whereas Flt3-CD150+CD48+ and Flt3-CD150-CD48+ LSK cells represent more differentiated multipotent progenitors (MPP1 and MPP2) without long-term engrafting capacity. Despite extensive investigation into BCR-ABL induced leukemogenesis, the impact of BCR-ABL expression on LSK subpopulations and the specific subpopulation with leukemia-initiating capacity remain unknown. Targeted expression of the BCR-ABL gene in murine hematopoietic stem and progenitor cells (HSPC), using a Tet-regulated SCL promoter, results in development of a chronic phase CML-like disorder (Blood 105:.324, 2005). Mice consistently develop leukocytosis, splenomegaly and expansion of bone marrow (BM) myeloid progenitors and primitive LSK cells following induction of BCR-ABL expression. Here we employed the SCL-tTA-BCR/ABL mouse model to investigate the effect of BCR-ABL expression on HSPC populations. BCR/ABL expression resulted in a 3-fold increase in granulocyte-macrophage progenitors (GMP) and a 1.5-fold increase in LSK cell numbers compared with non-induced controls, whereas numbers of common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP) were reduced. Despite expansion of total LSK cells, the number of LT-HSC was markedly reduced in the BM of BCR-ABL expressing mice (610±246 vs. 4,038±982). In contrast, an increase in MPP1 (6,150±1,813 vs. 3,185±1,247) and MPP2 (39,580±14,079 vs. 25,115±7,090) was seen. BCR-ABL mRNA expression was confirmed in each population by RT-PCR, with highest levels of expression seen in MPP cells. In vivo EdU labeling demonstrated increased cycling of LSK cells from BCR/ABL expressing mice compared to controls. We observed a vast increase in the number of GMP (11-fold), CMP (10-fold), MPP (4.5 fold) and LT-HSC (2.5 fold) in the spleen of BCR/ABL mice compared to controls. Since the functional potential of HSPC cannot be determined solely on the basis of cell surface markers, we also studied the ability of transplanted populations to generate leukemia in recipient mice. SCL-tTA/BCR-ABL transgenic mice were crossed with GFP transgenic mice to facilitate tracking of transplanted cells. Only LSK cells, but not CMP or GMP, from BM and spleen of BCR-ABL expressing mice were capable of generating CML-like disease and long term engraftment (>16 weeks) in recipient mice. The leukemic phenotype of donors was recapitulated in recipient mice; and leukemia could be transplanted to secondary and tertiary recipients. Further analysis revealed that the subpopulation of cells with a LT-HSC phenotype (Flt3-CD150+CD48-) within the LSK population was capable of generating CML-like disease and long term engraftment in recipient mice. Consistent with the diminished LT-HSC numbers demonstrated by flow cytometry, the frequency of functional HSC within the LSK population, as measured by competitive repopulation limiting dilution assays, was reduced in BCR-ABL expressing mice (1 in 234) compared to control mice (1 in 14). Interestingly, not all transplanted mice with long-term engrafted BCR-ABL-expressing cells developed leukemia. We determined that 1 in 6 Flt3-CD150+CD48- LSK cells possessed repopulation activity, whereas only 1 in 80 cells was capable of initiating leukemia in transplanted mice within 20 weeks, indicating that only a subset of BCR-ABL+ cells with long-term repopulating potential has leukemia-initiating capacity. In summary, BCR-ABL expression is associated with significant reduction in LT-HSC and expansion of MPP and GMP in the BM, and a marked increase in LT-HSC, MPP, CMP and GMP in the spleen of transgenic mice. Reduced LT-HSC numbers in BM may be explained by increased proliferation of BCR-ABL-expressing HSC and their enhanced egress from the BM to extramedullary locations such as the spleen. BCR-ABL expressing LT-HSC demonstrated long term engraftment and secondary transplantation capacity. However, only a fraction of BCR-ABL-expressing long-term repopulating cells has leukemia-initiating capacity, suggesting that additional cell intrinsic or extrinsic factors besides BCR-ABL expression may play a role in determining their leukemogenic potential.