Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3⁻ AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL).1,2 Here, we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG are hallmarks of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases, and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt utilizes a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivating domains of ERG, but inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia, in which DUX4 deregulation results in loss-of-function of ERG, either by deletion or induction of expression of an isoform that is a dominant negative inhibitor of wild type ERG function.

Hematopoietic stem cells (HSCs) reside in a bone marrow niche in a nondividing state from which they occasionally are aroused to undergo cell division. Yet, the mechanism underlying this unique feature remains largely unknown. We have recently shown that freshly isolated CD34 ؊ KSL hematopoietic stem cells (HSCs) in a hibernation state exhibit inhibited lipid raft clustering. Lipid raft clustering induced by cytokines is essential for HSCs to augment cytokine signals to the level enough to re-enter the cell cycle. Here we screened candidate niche signals that inhibit lipid raft clustering, and identified that transforming growth factor-␤ (TGF-␤) efficiently inhibits cytokine-mediated lipid raft clustering and induces HSC hibernation ex vivo. Smad2 and Smad3, the signaling mol- IntroductionDormancy or hibernation of hematopoietic stem cells (HSCs), which is indispensable for HSC maintenance, is known to occur solely in the particular bone marrow (BM) microenvironment known as the HSC niche. Most of the HSCs are in the G 0 phase in the BM niche. However, HSCs are recruited into the cell cycle at long intervals, on average every 1 to 2 months. 1,2 Thus, the capacity to enter and to leave a hibernation-like state is one of the properties of "stemness." The so-called stromal cells in the HSC BM niche, including osteoblasts, fibroblasts, adipocytes, and endothelial cells, produce several secreted and membrane-bound growth factors. 3 Several signaling pathways have been characterized that keep HSCs in hibernation or undifferentiated states. These include the Ang-1-Tie-2 signal, 4 the Notch ligand-Notch signal, 5 the Ncadherin homotypic signal, 6 and the transforming growth factor-␤ (TGF-␤) signal. 7 However, the precise molecular mechanisms underlying HSC hibernation remain largely elusive.Mouse BM HSCs are enriched exclusively in CD34 Ϫ c-Kit ϩ Sca-1 ϩ lineage marker-negative (Lin Ϫ ) (CD34 Ϫ KSL) cells, a population representing 0.004% of BM mononuclear cells, whereas CD34 ϩ KSL cells are progenitors with short-term repopulating capacity. 8 We have recently reported that HSCs use the PI3K-AktFoxO signaling pathway to regulate their hibernation state, as does C elegans in dauer formation. 9 Akt is inactive in the cytoplasm of freshly isolated hibernating CD34 Ϫ KSL HSCs, and FoxOs, its downstream targets, are active in their nuclei. In contrast, Akt is active in cycling CD34 ϩ KSL progenitors and phosphorylated FoxOs are excluded to the cytoplasm. Of note is our discovery that lipid raft status finely tunes cytokine signal levels and regulates Akt activity. Lipid raft microdomains are cholesterol-and glycosphingolipid-enriched patches in the plasma membrane into which various functional molecules are distributed. Lipid rafts act as platforms for cellular functions that include cytokine signaling, membrane trafficking, and cytoskeleton organization. 10 Because larger rafts have greater potential for concentration of transducers and for exclusion of negative regulators, lipid raft size controls signal intensity and fun...

Signaling mechanisms underlying self-renewal of leukemic stem cells (LSCs) are poorly understood, and identifying pathways specifically active in LSCs could provide opportunities for therapeutic intervention. T-cell immunoglobin mucin-3 (TIM-3) is expressed on the surface of LSCs in many types of human acute myeloid leukemia (AML), but not on hematopoietic stem cells (HSCs). Here, we show that TIM-3 and its ligand, galectin-9 (Gal-9), constitute an autocrine loop critical for LSC self-renewal and development of human AML. Serum Gal-9 levels were significantly elevated in AML patients and in mice xenografted with primary human AML samples, and neutralization of Gal-9 inhibited xenogeneic reconstitution of human AML. Gal-9-mediated stimulation of TIM-3 co-activated NF-κB and β-catenin signaling, pathways known to promote LSC self-renewal. These changes were further associated with leukemic transformation of a variety of pre-leukemic disorders and together highlight that targeting the TIM-3/Gal-9 autocrine loop could be a useful strategy for treating myeloid leukemias.