: To determine and directly compare the clinical course of white and Asian patients with paroxysmal nocturnal hemoglobinuria (PNH), data were collected for epidemiologic analysis on 176 patients from Duke University and 209 patients from Japan. White patients were younger with significantly more classical symptoms of PNH including thrombosis, hemoglobinuria, and infection, while Asian patients were older with more marrow aplasia. The mean fraction of CD59-negative polymorphonuclear cells (PMN) at initial analysis was higher among Duke patients than Japanese patients. In both cohorts, however, a larger PNH clone was associated with classical PNH symptoms, while a smaller PNH clone was associated with marrow aplasia. Thrombosis was significantly more prevalent in white patients than Asian patients, and was associated with a significantly higher proportion of CD59-negative PMN. For individual patients, CD59-negative populations varied considerably over time, but a decreasing PNH clone portended hematopoietic failure. Survival analysis revealed a similar death rate in each group, although causes of death were different and significantly more Duke patients died from thrombosis. Japanese patients had a longer mean survival time (32.1 yr vs. 19.4 yr), although Kaplan-Meier survival curves were not significantly different. Poor survival in both groups was associated with age over 50 years, severe leukopenia/neutropenia at diagnosis, and severe infection as a complication; additionally, thrombosis at diagnosis or follow-up for Duke patients and renal failure for Japanese patients were poor prognostic factors. These data identify important differences between white and Asian patients with PNH. Identification of prognostic factors will help the design of prospective clinical trials for PNH.

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.

Activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway and inactivation of wild-type p53 by murine double minute 2 homologue (Mdm2) overexpression are frequent molecular events in acute myeloid leukemia (AML). We investigated the interaction of PI3K/Akt/mTOR and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 and the Mdm2 inhibitor Nutlin-3. We found that PI-103, which itself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformational change and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3 combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability. We suggest that PI-103 actively enhances downstream p53 signaling and that a combination strategy aimed at inhibiting PI3K/Akt/mTOR signaling and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (El/miR-125b-TG mice). El/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the El/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a proapoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells.

Summary. Among acquired stem cell disorders, pathological links between myelodysplastic syndromes (MDS) and aplastic anaemia (AA), and paroxysmal nocturnal haemoglobinuria (PNH) and AA, have been often described, whereas the relationship between MDS and PNH is still unclear. We analysed blood cells of patients with MDS to determine the incidence of the PNH clone, and analysed the PIG-A gene to find mutations characteristic of the PNH clone in MDS. In four (10%) of 40 patients with MDS, flow cytometry showed affected erythrocytes and granulocytes negative for decayaccelerating factor (DAF) and CD59. The population of affected erythrocytes was smaller in MDS patients with PNH clone (MDS/PNH) than in patients with de novo PNH, and haemolysis was milder in the MDS/PNH patients. PIG-A mutations were found in granulocytes of all patients with MDS/PNH. In type and site, the PIG-A mutations were heterogenous, similar to that observed in de novo PNH; i.e. no mutation specific to MDS/PNH was identified. Of note, three of four patients with MDS/PNH each had two PNH clones with different PIG-A mutations, suggesting that PIG-A is mutable in patients with MDS/PNH. In a MDS/PNH patient with trisomy 8, FISH detected a distinct karyotype in a portion of granulocytes with PNH phenotype, indicating that PNH and MDS partly shared affected cells. Thus, MDS predisposes to PNH by creating conditions favourable to the genesis of PNH clone. Considering the increasing prevalence and incidence of MDS, these disorders could be useful for investigating the mechanism by which PIG-A mutation is induced.