GATA2 regulates the erythropoietin receptor in t(12;21) ALL.

The t(12;21) (p13;q22) chromosomal translocation resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality in children with acute lymphoblastic leukemia (ALL). The erythropoietin receptor (EPOR), usually associated with erythroid progenitor cells, is highly expressed in ETV6/RUNX1 positive cases compared to other B-lineage ALL subtypes. Gene expression analysis of a microarray database and direct quantitative analysis of patient samples revealed strong correlation between EPOR and GATA2 expression in ALL, and higher expression of GATA2 in t(12;21) patients. The mechanism of EPOR regulation was mainly investigated using two B-ALL cell lines: REH, which harbor and express the ETV6/RUNX1 fusion gene; and NALM-6, which do not. Expression of EPOR was increased in REH cells compared to NALM-6 cells. Moreover, of the six GATA family members only GATA2 was differentially expressed with substantially higher levels present in REH cells. GATA2 was shown to bind to the EPOR 5'-UTR in REH, but did not bind in NALM-6 cells. Overexpression of GATA2 led to an increase in EPOR expression in REH cells only, indicating that GATA2 regulates EPOR but is dependent on the cellular context. Both EPOR and GATA2 are hypomethylated and associated with increased mRNA expression in REH compared to NALM-6 cells. Decitabine treatment effectively reduced methylation of CpG sites in the GATA2 promoter leading to increased GATA2 expression in both cell lines. Although Decitabine also reduced an already low level of methylation of the EPOR in NALM-6 cells there was no increase in EPOR expression. Furthermore, EPOR and GATA2 are regulated post-transcriptionally by miR-362 and miR-650, respectively. Overall our data show that EPOR expression in t(12;21) B-ALL cells, is regulated by GATA2 and is mediated through epigenetic, transcriptional and post-transcriptional mechanisms, contingent upon the genetic subtype of the disease.

Inhibition of mitochondrial aconitase by succination in fumarate hydratase deficiency.

The gene encoding the Krebs cycle enzyme fumarate hydratase (FH) is mutated in hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity causes accumulation of intracellular fumarate, which can directly modify cysteine residues to form 2-succinocysteine through succination. We undertook a proteomic-based screen in cells and renal cysts from Fh1 (murine FH)-deficient mice and identified 94 protein succination targets. Notably, we identified the succination of three cysteine residues in mitochondrial Aconitase2 (ACO2) crucial for iron-sulfur cluster binding. We show that fumarate exerts a dose-dependent inhibition of ACO2 activity, which correlates with increased succination as determined by mass spectrometry, possibly by interfering with iron chelation. Importantly, we show that aconitase activity is impaired in FH-deficient cells. Our data provide evidence that succination, resulting from FH deficiency, targets and potentially alters the function of multiple proteins and may contribute to the dysregulated metabolism observed in HLRCC.

HOXA/PBX3 knockdown impairs growth and sensitizes cytogenetically normal acute myeloid leukemia cells to chemotherapy.

The cytogenetically normal subtype of acute myeloid leukemia is associated with an intermediate risk which complicates therapeutic options. Lower overall HOX/TALE expression appears to correlate with more favorable prognosis/better response to treatment in some leukemias and solid cancer. The functional significance of the associated gene expression and response to chemotherapy is not known. Three independent microarray datasets obtained from large cohorts of patients along with quantitative polymerase chain reaction validation were used to identify a four-gene HOXA/TALE signature capable of prognostic stratification. Biochemical analysis was used to identify interactions between the four encoded proteins and targeted knockdown used to examine the functional importance of sustained expression of the signature in leukemia maintenance and response to chemotherapy. An 11 HOXA/TALE code identified in an intermediate-risk group of patients (n=315) compared to a group with a favorable risk (n=105) was reduced to a four-gene signature of HOXA6, HOXA9, PBX3 and MEIS1 by iterative analysis of independent platforms. This signature maintained the favorable/intermediate risk partition and where applicable, correlated with overall survival in cytogenetically normal acute myeloid leukemia. We further showed that cell growth and function are dependent on maintained levels of these core genes and that direct targeting of HOXA/PBX3 sensitizes cytogenetically normal acute myeloid leukemia cells to standard chemotherapy. Together the data support a key role for HOXA/TALE in cytogenetically normal acute myeloid leukemia and demonstrate that targeting of clinically significant HOXA/PBX3 elements may provide therapeutic benefit to patients with this subtype of leukemia.

Editorial: Our top 10 developments in stem cell biology over the last 30 years.

To celebrate 30 years of peer-reviewed publication of cutting edge stem cell research in Stem Cells, the first journal devoted to this promising field, we pause to review how far we have come in the three-decade lifetime of the Journal. To do this, we will present our views of the 10 most significant developments that have advanced stem cell biology where it is today. With the increasing rate of new data, it is natural that the bulk of these developments would have occurred in recent years, but we must not think that stem cell biology is a young science. The idea of a stem cell has actually been around for quite a long time having appeared in the scientific literature as early as 1868 with Haeckels' concept of a stamzelle as an uncommitted or undifferentiated cell responsible for producing many types of new cells to repair the body [Naturliche Schopfungsgeschichte, 1868; Berlin: Georg Reimer] but it took many years to obtain hard evidence in support of this theory. Not until the work of James Till and Ernest McCulloch in the 1960s did we have proof of the existence of stem cells and until the derivation of embryonal carcinoma cells in the 1960s-1970s and the first embryonic stem cell in 1981, such adult or tissue-specific stem cells were the only known class. The first issue of Stem Cells was published in 1981; no small wonder that most of its papers were devoted to hematopoietic progenitors. More recently, induced pluripotent stem cells (iPSCs) have been developed, and this is proving to be a fertile area of investigation as shown by the volume of publications appearing not only in Stem Cells but also in other journals over the last 5 years. The reader will note that many of the articles in this special issue are concerned with iPSC; however, this reflects the current surge of interest in the topic rather than any deliberate attempt to ignore other areas of stem cell investigation.

Erythropoietin-induced activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK pathways promotes malignant cell behavior in a modified breast cancer cell line.

Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

The worldwide distribution of the VHL 598C>T mutation indicates a single founding event.

The first congenital defect of hypoxia-sensing homozygosity for VHL 598C>T mutation was recently identified in Chuvash polycythemia. Subsequently, we found this mutation in 11 unrelated individuals of diverse ethnic backgrounds. To address the question of whether the VHL 598C>T substitution occurred in a single founder or resulted from recurrent mutational events in human evolution, we performed haplotype analysis of 8 polymorphic markers covering 340 kb spanning the VHL gene on 101 subjects bearing the VHL 598C>T mutation, including 72 homozygotes (61 Chuvash and 11 non-Chuvash) and 29 heterozygotes (11 Chuvash and 18 non-Chuvash), and 447 healthy unrelated individuals from Chuvash and other ethnic groups. The differences in allele frequencies for each of the 8 markers between 447 healthy controls (598C) and 101 subjects bearing the 598T allele (P < 10(-7)) showed strong linkage disequilibrium. Haplotype analysis indicated a founder effect. We conclude that the VHL 598C>T mutation, the most common defect of congenital polycythemia yet found, was spread from a single founder 1,000 to 62,000 years ago.

The cellular biology of erythropoietin receptors.

Long thought to act only as a hormone that was the primary regulator of red blood cell production, erythropoietin is now known to have a whole spectrum of activity. Organs such as the brain, ovary, oviduct, uterus, and testis have erythropoietin receptors. Because erythropoietin receptors exist in the paracrine and autocrine systems, as well as the hormonal system, the beneficial effects of administering human erythropoietin are likely to extend beyond its effect in raising hematocrit. It may even be possible in the future to use the receptors to target a drug to a tumor without damaging the surrounding tissue.