Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34 + /CD38 − or CD34 + /HLA-DR − . Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3R␣ or CD123) was strongly expressed in CD34 + /CD38 − cells (98 ± 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34 + /CD38 − cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3R␣ positive cells, purified CD34 + /CD123 + leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123 + cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.

A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax‐related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX–ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX–ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin‐3 and stem cell factor. Immortalized cells contained structurally intact HRX–ENL proviral DNA and expressed a low‐level of HRX–ENL mRNA. In contrast, wild‐type ENL or a deletion mutant of HRX–ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX–ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX–ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain‐of‐function mechanism for HRX–ENL‐mediated leukemogenesis.

Acute myelogenous leukemia (AML) is typically a disease of stem͞ progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G 0 cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-L-leucyl-L-leucyl-L-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominantnegative allele of inhibitor of nuclear factor B (I B␣) demonstrate that inhibition of nuclear factor B (NF-B) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis.Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.