Curt I. Civin

The translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein

The recurrent translocation t(8;16)(p11 ;p13) is a cytogenetic hallmark for the M4/M5 subtype of acute myeloid leukaemia. Here we identify the breakpoint-associated genes. Positional cloning on chromosome 16 implicates the CREB-binding protein (CBP), a transcriptional adaptor/coactivator protein. At the chromosome 8 breakpoint we identify a novel gene, MOZ, which encodes a 2,004-amino-acid protein characterized by two C4HC3 zinc fingers and a single C2HC zinc finger in conjunction with a putative acetyltransferase signature. In-frame MOZ–CBP fusion transcripts combine the MOZ finger motifs and putative acetyltransferase domain with a largely intact CBP. We suggest that MOZ may represent a chromatin-associated acetyltransferase, and raise the possibility that a dominant MOZ–CBP fusion protein could mediate leukaemogenesis via aberrant chromatin acetylation.

CD34+ hematopoietic stem-progenitor cell microRNA expression and function: A circuit diagram of differentiation control

MicroRNAs (miRNAs) are a recently identified class of epigenetic elements consisting of small noncoding RNAs that bind to the 3′ untranslated region of mRNAs and down-regulate their translation to protein. miRNAs play critical roles in many different cellular processes including metabolism, apoptosis, differentiation, and development. We found 33 miRNAs expressed in CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human bone marrow and mobilized human peripheral blood stem cell harvests. We then combined these data with human HSPC mRNA expression data and with miRNA-mRNA target predictions, into a previously undescribed miRNA:mRNA interaction database called the Transcriptome Interaction Database. The in silico predictions from the Transcriptome Interaction Database pointed to miRNA control of hematopoietic differentiation through translational control of mRNAs critical to hematopoiesis. From these predictions, we formulated a model for miRNA control of stages of hematopoiesis in which many of the genes specifying hematopoietic differentiation are expressed by HSPCs, but are held in check by miRNAs until differentiation occurs. We validated miRNA control of several of these target mRNAs by demonstrating that their translation in fact is decreased by miRNAs. Finally, we chose miRNA-155 for functional characterization in hematopoiesis, because we predicted that it would control both myelopoiesis and erythropoiesis. As predicted, miRNA-155 transduction greatly reduced both myeloid and erythroid colony formation of normal human HSPCs.

Mobile Interspersed Repeats Are Major Structural Variants in the Human Genome

Characterizing structural variants in the human genome is of great importance, but a genome wide analysis to detect interspersed repeats has not been done. Thus, the degree to which mobile DNAs contribute to genetic diversity, heritable disease, and oncogenesis remains speculative. We perform transposon insertion profiling by microarray (TIP-chip) to map human L1(Ta) retrotransposons (LINE-1 s) genome-wide. This identified numerous novel human L1(Ta) insertional polymorphisms with highly variant allelic frequencies. We also explored TIP-chips usefulness to identify candidate alleles associated with different phenotypes in clinical cohorts. Our data suggest that the occurrence of new insertions is twice as high as previously estimated, and that these repeats are under-recognized as sources of human genomic and phenotypic diversity. We have just begun to probe the universe of human L1(Ta) polymorphisms, and as TIP-chip is applied to other insertions such as Alu SINEs, it will expand the catalog of genomic variants even further.

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: the significance of the stem‐cell glycoprotein CD34 (My 10)

A series of 23 monoclonal antibodies reactive with normal lymphoid and myeloid cells at various stages of differentiation were used to characterize 96 adult patients with acute myelocytic leukaemia (AML), concentrating on the possible role the expression of these antigens may have in predicting response to intensive chemotherapy. Only the expression of CD34 (P= 0·008) and HLA‐DR (P= 0·035) was significant in predicting response to therapy; patients with leukaemic cells expressing CD34 (My10) had a complete remission (CR) rate of 59% compared to 87% for those with blasts not expressing the antigen. In a multivariate analysis predicting for CR, the expression of CD34, the disease category (de novo AML versus secondary AML [SAML] or a history of antecedent haematological disorder [AHD]), and WBC were significant covariates. Adjusting for disease category and WBC, patients with CD34‐positive AML were one‐third as likely to enter CR as with those with disease not expressing the antigen (P= 0·066). Comparison of clinical characteristics between the 58 patients whose leukaemia expressed CD34 and the 33 which were CD34‐negative found that patients with CD34‐positive AML had a higher incidence of SAML and AHD, a lower WBC at diagnosis, and a more frequent incidence of chromosomal abnormalities involving chromosomes 5 and/or 7. Twenty‐eight of these patients also had immunophenotyping performed at relapse. Patients who presented with CD34‐positive AML, and entered remission, and then relapsed all recurred with CD34‐positive leukaemia; there was no case of CD34‐positive AML at diagnosis relapsing with CD34‐negative disease. In addition, there were patients presenting with CD34‐negative AML and then relapsing with CD34‐positive AML. These results suggest that intensive cytoreductive therapy is ineffective against CD34‐positive AML. Patients who present with CD34‐positive AML may require different therapeutic approaches to completely eradicate their disease.

Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice

OBJECTIVE For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development. MATERIALS AND METHODS We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures. RESULTS We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+). CONCLUSIONS Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.

Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4−/− fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4+/+ cells in competitive repopulation assays. However, hematopoietic “KLF4−/− chimeras” generated by transplantation of KLF4−/− fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115+Gr1+) monocytes, and had reduced numbers of resident (CD115+Gr1−) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4−/− chimeras, bone marrow monocytic cells from KLF4−/− chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

Pediatric Oncology Group (POG) studies of acute myeloid leukemia (AML): a review of four consecutive childhood AML trials conducted between 1981 and 2000.

From 1981 to 2000, a total of 1823 children with acute myeloid leukemia (AML) enrolled on four consecutive Pediatric Oncology Group (POG) clinical trials. POG 8101 demonstrated that the induction rate associated with the 3+7+7 combination of daunorubicin, Ara-C, and 6-thioguanine (DAT) was greater than that associated with an induction regimen used to treat acute lymphoblastic leukemia (82 vs 61%; P=0.02). Designed as a pilot study to determine the feasibility of administration of noncross-resistant drug pairs and later modified to assess the effect of dose intensification of Ara-C during the second induction course, POG 8498 confirmed the high initial rate of response to DAT (84.2%) and showed that dose intensification of Ara-C during the second induction course resulted in a trend toward higher event-free survival (EFS) estimates than did standard-dose DAT (2+5) during the second induction course (5 year EFS estimates, 22 vs 27%; P=0.33). Age <2 years and leukocyte count <100 000/mm3 emerged as significantly good prognostic factors. The most significant observation made in the POG 8498 study was the markedly superior outcome of children with Downs syndrome who were treated on the high-dose Ara-C regimen. POG 8821 compared the efficacy of autologous bone marrow transplantation (BMT) with that of intensive consolidation chemotherapy. Intent-to-treat analysis revealed similar 5-year EFS estimates for the group that underwent autologous BMT (36±4.7%) and for the group that received only intensive chemotherapy (35±4.5%) (P=0.25). There was a high rate of treatment-related mortality in the autologous transplantation group. The study demonstrated superior results of allogeneic BMT for patients with histocompatible related donors (5-year EFS estimate 63±5.4%) and of children with Downs syndrome (5-year EFS estimate, 66±8.6%). The POG 9421 AML study evaluated high-dose Ara-C as part of the first induction course and the use of the multidrug resistance modulator cyclosporine. Preliminary results showed that patients receiving both high-dose Ara-C for remission induction and the MDR modulator for consolidation had a superior outcome (5-year EFS estimate, 42±8.2%) than did patients receiving other treatment; however, the difference was not statistically significant. These four studies demonstrate the importance of dose intensification of Ara-C in the treatment of childhood AML; cytogenetics as the single most prognostic factor and the unique curability of AML in children with Downs syndrome.

Highly purified CD34-positive cells reconstitute hematopoiesis

PURPOSE The objective of this study was to characterize CD34+ cell grafts, obtained using a novel technique, from children undergoing autologous bone marrow transplantation (BMT) for cancer therapy. In particular, we wanted to determine if the CD34+ marrow cell grafts generated hematopoietic reconstitution, since a positive result would motivate further development and use of this methodology. PATIENTS AND METHODS This pilot feasibility clinical trial involved 13 patients < or = 25 years of age with advanced solid tumors, including seven children with neuroblastoma. Harvested bone marrow underwent immunomagnetic CD34+ selection. RESULTS In three of 13 enrolled patients, low purities of the CD34+ preparations disqualified the use of the CD34+ marrow grafts. Ten patients received myeloablative chemotherapy with etoposide, carboplatin, and cyclophosphamide, then were transplanted with CD34+ marrow grafts. In the 10 patients transplanted with CD34(+)-selected cells, the CD34+ cell purity (nucleated RBCs excluded) in the cell graft preparation was 91% total cell recovery from the starting light-density cells 2.2%, CD34+ cell recovery 38%, colony-forming unit-granulocyte-macrophage (CFU-GM) recovery 23%, and estimated tumor-cell depletion 2.6 logs (medians). The CD34+ marrow grafts administered to these patients contained a median of 2.3 x 10(6) nucleated cells, 1.4 x 10(6) CD34+ cells, and 1.3 x 10(4) CFU-GM per kilogram patient weight. Most patients experienced only the toxicities previously observed with this myeloblative chemotherapy regimen, although two unusual toxicities were observed. All 10 patients transplanted with CD34+ cell grafts engrafted. CONCLUSION The CD34+ purified grafts were enriched in stem/progenitor cells, with five of these 10 preparations containing > or = 94% CD34+ cells. Engraftment with CD34(+)-purified cell grafts as pure as 99% confirms that autologous CD34+ cells, alone, are sufficient to provide hematopoietic rescue for myeloablated patients. The best purification results were obtained on small marrow harvests from patients with neuroblastoma. The engraftment of highly purified CD34+ cells obtained by this technology and the antitumor effect of the transplant, by which two of 10 poor prognosis patients remain clinically free of tumor, have stimulated further clinical trials.

Lentiviral vectors with two independent internal promoters transfer high-level expression of multiple transgenes to human hematopoietic stem-progenitor cells

Lentiviral vectors (LVs) offer several advantages over traditional oncoretroviral vectors. LVs efficiently transduce slowly dividing cells, including hematopoietic stem-progenitor cells (HSCs), resulting in stable gene transfer and expression. Additionally, recently developed self-inactivating (SIN) LVs allow promoter-specific transgene expression. For many gene transfer applications, transduction of more than one gene is needed. We obtained inconsistent results in our attempts to coexpress two transgenes linked by an internal ribosomal entry site (IRES) element in a single bicistronic LV transcript. In more than six bicistronic LVs we constructed containing a gene of interest followed by an IRES and the GFP reporter gene, GFP fluorescence was undetectable in transduced cells. We therefore investigated how to achieve consistent and efficient coexpression of two transgenes by LVs. In a SIN LV containing the elongation factor 1alpha promoter, we included a second promoter from cytomegalovirus, the phosphoglycerate kinase gene, or the HLA-DRalpha gene. Using a single LV containing two constitutive promoters, we achieved strong and sustained expression of both transgenes in transduced engrafting CD34(+) HSCs and their progeny, as well as in other human cell types. Thus, such dual-promoter LVs can coexpress multiple transgenes efficiently in a single target cell and will enable many gene transfer applications.