The malignant clone in acute myeloid leukemia (AML) is organized hierarchically with

  • more mature cells programmed to undergo natural apoptosis after a variable number of cell divisions and

  • immature primitive cells that have self-renewal and NOD/SCID mouse-repopulating capacity. In most types of AML, leukemic stem cells supposedly reside within the CD34+/CD38− fraction of the leukemic clone.

Because of their long term leukemia-repopulating capacity, AML stem cells are a logic target of therapy. However, so far, little is known about the regulation of growth and survival of these cells. We examined the expression of cytokine receptors (SCFR/KIT, IL-3Rα, GM-CSFRα, IL-3/GM-CSFRβ, G-CSFR, M-CSFR, TGFβR/endoglin, EPOR, TPOR/MPL, FLT3, VEGFR/KDR) and of other molecular targets and markers (CD33, CD44, CD133) on CD34+/CD38− cells in patients with AML (n=30), and determined responses to cytokine-ligands, conventional antileukemic drugs (ARA-C, fludarabine) or targeted drugs (Gemtuzumab/Ozogamicin, GO = Mylotarg®) in these cells. Apoptosis in AML stem cells was analyzed by combined staining for surface markers and AnnexinV. In a group of patients, CD34+/CD38- cells were purified to homogeneity by cell sorting and examined for 3H-thymidine uptake. Unexpectedly, AML stem cells were found to display a highly variable pattern of cytokine receptors and cell surface targets. In fact, only the IL-3R and CD44 were expressed consistently on all AML stem cells in all donors tested. In most patients, at least a subset of AML stem cells also co-expressed the SCFR/KIT, G-CSFR, TGFβR, FLT3, CD33, and CD133. By contrast, AML stem cells in most donors were found to lack substantial amounts of the GM-CSFR, M-CSFR, EPOR, TPOR, and VEGFR/KDR. When cultured in RPMI-1640 medium plus 10% FCS, about 5–15% of the CD34+/CD38- cells and about 10–40% of the more mature CD34+ AML cells were found to undergo spontaneous apoptosis within 48 hours. Spontaneous apoptosis was prevented by exposure to SCF, IL-3, or G-CSF, but not by exposure to EPO. ARA-C (0.5–5 μg/ml), fludarabine (0.1–5 μg/ml), and GO/Mylotarg® (1 μg/ml) were found to promote apoptosis in CD34+/CD38− cells and in more mature AML cells in all donors tested. The effects of ARA-C and fludarabine on AML cells were found to be dose-dependent. Moreover, we were able to show that ARA-C (0.5 μg/ml) and fludarabine (0.1 μg/ml) cooperate with each other in producing apoptosis in AML (stem) cells. 3H-thymidine uptake experiments performed on purified CD34+/CD38− AML stem cells confirmed the growth-inhibitory effects of these drugs. In particular, ARA-C and fludarabine were found to inhibit the cytokine-induced 3H-thymidine uptake in sorted AML stem cells in all donors examined. In summary, our data show that multi-color flow cytometry and combined staining for surface markers and AnnexinV is a powerful approach to determine apoptosis-preventing effects of cytokines and apoptosis-inducing effects of anti-leukemic drugs in immature CD34+/CD38− AML progenitor cells. Using this assay, it should be possible to identify combinations of targeted and/or conventional drugs eliminating maximal numbers of leukemic stem cells in AML.

Disclosures: Holmes:Domantis Limited: Employment. Valent:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding.

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