T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Abstract 3980

Poster Board III-916

Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin^-/Sca1⁺/c-Kit⁺/Flk2^-) and ST-HSC (lin^-/Sca1⁺/c-Kit⁺/Flk2⁺). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin^-/Sca1⁺/c-Kit⁺/Flk2^- population but not from that transplanted with lin^-/Sca1⁺/c-Kit⁺/Flk2⁺ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model.