Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.

Human NK cells are divided into CD56brightCD16− cells and CD56dimCD16+ cells. We tested the hypothesis that CD56bright NK cells can differentiate into CD56dim cells by prospectively isolating and culturing each NK subset in vitro and in vivo. Our results show that CD56bright cells can differentiate into CD56dim both in vitro, in the presence of synovial fibroblasts, and in vivo, upon transfer into NOD-SCID mice. In vitro, this differentiation was inhibited by fibroblast growth factor receptor-1 Ab, demonstrating a role of the CD56 and fibroblast growth factor receptor-1 interaction in this process. Differentiated CD56dim cells had reduced IFN-γ production but increased perforin expression and cytolysis of cell line K562 targets. Flow cytometric fluorescent in situ hybridization demonstrated that CD56bright NK cells had longer telomere length compared with CD56dim NK cells, implying the former are less mature. Our data support a linear differentiation model of human NK development in which immature CD56bright NK cells can differentiate into CD56dim cells.

Residence of cancer-propagating cells (CPCs) within preferential microenvironmental niches has a major part in evading therapy. However, the nature of niches involved and the mechanisms protecting CPCs remain largely unknown. We addressed these issues in mouse transplantation models of acute lymphoblastic leukemia (ALL). When the engrafted leukemic cells substantially damaged adjacent microenvironment in the bone marrow (BM), after chemotherapy small foci of CPCs were retained, surrounded by sheaths of supporting cells that comprise a protective niche. We investigated patients' BM biopsies and found evidence of a similar process in patients receiving induction therapy. The efficacy of chemotherapy was enhanced by interfering with the niche formation or function. We therefore identified a therapy-induced niche that protects CPCs.

Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-β. This facilitates the competitive expansion of TEL-AML1-expressing cells in the presence of TGF-β. Further analysis indicated that TEL-AML1 binds to a principal TGF-β signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-β-mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34 + CD38 -CD19 + ) and a marked growth advantage in the presence of TGF-β. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1-expressing preleukemic clones.

Evidence suggests the transcription factor GATA-2 is a critical regulator of murine hematopoietic stem cells. Here, we explore the relation between GATA-2 and cell proliferation and show that inducing GATA-2 increases quiescence (

Clinically successful hematopoietic cell transplantation is dependent on hematopoietic stem and progenitor cells. Here we identify the matricellular protein Nephroblastoma Overexpressed (Nov, CCN3) as being essential for their functional integrity. Nov expression is restricted to the primitive (CD34) compartments of umbilical vein cord blood, and its knockdown in these cells by lentivirus-mediated RNA interference abrogates their function in vitro and in vivo. Conversely, forced expression of Nov and addition of recombinant Nov protein both enhance primitive stem and/or progenitor activity. Taken together, our results identify Nov (CCN3) as a regulator of human hematopoietic stem or progenitor cells.

Background AML1/RUNX1 is the most frequently mutated gene in leukaemia and is central to the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 isoforms within these primitive compartments is unclear. Here we investigate whether altering relative expression of AML1 isoforms impacts the balance between cell self-renewal and differentiation in vitro and in vivo.Methods and FindingsThe human AML1a isoform encodes a truncated molecule with DNA-binding but no transactivation capacity. We used a retrovirus-based approach to transduce AML1a into primitive haematopoietic cells isolated from the mouse. We observed that enforced AML1a expression increased the competitive engraftment potential of murine long-term reconstituting stem cells with the proportion of AML1a-expressing cells increasing over time in both primary and secondary recipients. Furthermore, AML1a expression dramatically increased primitive and committed progenitor activity in engrafted animals as assessed by long-term culture, cobblestone formation, and colony assays. In contrast, expression of the full-length isoform AML1b abrogated engraftment potential. In vitro, AML1b promoted differentiation while AML1a promoted proliferation of progenitors capable of short-term lymphomyeloid engraftment. Consistent with these findings, the relative abundance of AML1a was highest in the primitive stem/progenitor compartment of human cord blood, and forced expression of AML1a in these cells enhanced maintenance of primitive potential both in vitro and in vivo.ConclusionsThese data demonstrate that the “a” isoform of AML1 has the capacity to potentiate stem and progenitor cell engraftment, both of which are required for successful clinical transplantation. This activity is consistent with its expression pattern in both normal and leukaemic cells. Manipulating the balance of AML1 isoform expression may offer novel therapeutic strategies, exploitable in the contexts of leukaemia and also in cord blood transplantation in adults, in whom stem and progenitor cell numbers are often limiting.

Hypoxia-inducible factor 1 (HIF-1) is a crucial transcription factor for the cellular adaptive response to hypoxia, which contributes to multiple events in cancer biology. MicroRNAs (miRNAs) are involved in almost all cellular activities such as differentiation, proliferation, and apoptosis. In this work, we use miRNA microarrays to profile miRNA expression in acute myeloid leukemia (AML) cells with inducible HIF-1a expression, and identify 19 differentially expressed miRNAs. Our study shows that HIF-1a represses the expression of miR-17 and miR-20a by downregulating c-Myc expression. These two miRNAs alleviate hypoxia and HIF-1a-induced differentiation of AML cells. More intriguingly, miR-17 and miR-20a directly inhibit the p21 and STAT3 (signal transducer and activator of transcription 3) expression, both of which can reverse miR-17/miR-20a-mediated abrogation of HIF-1a-induced differentiation. Moreover, we show in vivo that miR-20a contributes to HIF-1a-induced differentiation of leukemic cells. Taken together, our results suggest that HIF-1a regulates the miRNA network to interfere with AML cell differentiation, representing a novel molecular mechanism for HIF-1-mediated anti-leukemic action. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcriptional factor that consists of the oxygen-sensitive alpha subunit (HIF-1a) and the constitutively expressed beta subunit (HIF-1b), is a master regulator for the cellular adaptive response to oxygen concentration. 1 Under normoxic conditions, proline residues 402 and 564 of the HIF-1a protein are hydroxylated by specific prolyl hydroxylases (PHDs) that utilize O 2 and a-ketoglutarate as co-factors. The hydroxylated HIF-1a protein is subject to ubiquitination by the E3 ubiquitin ligase von Hippel-Lindau (VHL), which leads to its degradation. In contrast, hypoxic conditions cause the accumulation of HIF-1a protein by inhibiting its hydroxylation, and subsequent ubiquitination and degradation. 2 The stabilized HIF-1a protein translocates into the nucleus, where it forms a heterodimer with HIF-1b and modulates the expression of hundreds of genes through binding to hypoxia-responsive elements (HREs; 5 0 -RCGTG-3 0 ) on their promoters. These HIF-1-targeted genes help the cell adapt to hypoxia by influencing processes such as erythropoiesis, angiogenesis, cell metabolism, growth, apoptosis, and differentiation.Intriguingly, HIF-1a has been shown to contribute to the pathogenesis and progression of multiple kinds of diseases, including cancer. 1,3 Although a hypoxic microenvironment is regarded as a hallmark of solid tumors, and hypoxia-stabilized HIF-1a protein contributes to tumor growth, angiogenesis, and metastasis, 4 several groups, including our own, have reported that HIF-1a protein can trigger acute myeloid leukemia (AML) cells to undergo differentiation through a transcription-independent mechanism, inhibiting the progression of AML. [5][6][7][8][9] MicroRNAs (miRNAs) are a distinct class of small noncoding RNAs of around 22 nucleotides in length that posttra...