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Aurora kinases play an important role in chromosome alignment,segregation,and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases,and ZM447439,a potent inhibitor of Aurora kinases,effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152,a highly selective inhibitor of Aurora B kinase,on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60,NB4,MOLM13),ALL line (PALL-2),biphenotypic leukemia (MV4-11),acute eosinophilic leukemia (EOL-1),and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM,as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis,as measured by cell-cycle analysis and annexin V staining,respectively. Of note,AZD1152 synergistically enhanced the antiproliferative activity of vincristine,a tubulin depolymerizing agent,and daunorubicin,a topoisomerase II inhibitor,against the MOLM13 and PALL-2 cells in vitro. Furthermore,AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together,AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation. View Publication

Overexpression of wild-type MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We evaluated whether MN1 plays a functional role in leukemogenesis. We demonstrate using retroviral gene transfer and bone marrow (BM) transplantation that MN1 overexpression rapidly induces lethal AML in mice. Insertional mutagenesis and chromosomal instability were ruled out as secondary aberrations. MN1 increased resistance to all-trans retinoic acid (ATRA)-induced cell-cycle arrest and differentiation by more than 3000-fold in vitro. The differentiation block could be released by fusion of a transcriptional activator (VP16) to MN1 without affecting the ability to immortalize BM cells,suggesting that MN1 blocks differentiation by transcriptional repression. We then evaluated whether MN1 expression levels in patients with AML (excluding M3-AML) correlated with resistance to ATRA treatment in elderly patients uniformly treated within treatment protocol AMLHD98-B. Strikingly,patients with low MN1 expression who received ATRA had a significantly prolonged event-free (P = .008) and overall (P = .04) survival compared with patients with either low MN1 expression and no ATRA,or high MN1 expression with or without ATRA. MN1 is a unique oncogene in hematopoiesis that both promotes proliferation/self-renewal and blocks differentiation,and may become useful as a predictive marker in AML treatment. View Publication

Protein geranylgeranyltransferase type I (GGTase-I) is responsible for the posttranslational lipidation of CAAX proteins such as RHOA,RAC1,and cell division cycle 42 (CDC42). Inhibition of GGTase-I has been suggested as a strategy to treat cancer and a host of other diseases. Although several GGTase-I inhibitors (GGTIs) have been synthesized,they have very different properties,and the effects of GGTIs and GGTase-I deficiency are unclear. One concern is that inhibiting GGTase-I might lead to severe toxicity. In this study,we determined the effects of GGTase-I deficiency on cell viability and K-RAS-induced cancer development in mice. Inactivating the gene for the critical beta subunit of GGTase-I eliminated GGTase-I activity,disrupted the actin cytoskeleton,reduced cell migration,and blocked the proliferation of fibroblasts expressing oncogenic K-RAS. Moreover,the absence of GGTase-I activity reduced lung tumor formation,eliminated myeloproliferative phenotypes,and increased survival of mice in which expression of oncogenic K-RAS was switched on in lung cells and myeloid cells. Interestingly,several cell types remained viable in the absence of GGTase-I,and myelopoiesis appeared to function normally. These findings suggest that inhibiting GGTase-I may be a useful strategy to treat K-RAS-induced malignancies. View Publication

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF),peripheral blood CD34+ cells isolated from patients with IMF,polycythemia vera (PV),and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is,therefore,likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9,therefore,likely contribute to the development of many pathological epiphenomena associated with IMF. View Publication

BACKGROUND: ALDH(br) cells express high aldehyde dehydrogenase (ALDH) activity and have progenitor cell activity in several contexts. We characterized human BM ALDH(br) cells to determine whether cell sorting based on ALDH activity isolates potentially useful populations for cell therapy. METHOD: We measured the expression of ALDH and cell-surface Ag by flow cytometry and compared the ability of sorted ALDH(br),and BM populations remaining after ALDH(br) cells were removed (ALDH(dim) populations),to develop into several cell lineages in culture. RESULTS: The ALDH(br) population comprised 1.2+/-0.8% (mean+/-SD,n=30) nucleated cells and was enriched in cells expressing CD34,CD117,CD105,CD127,CD133 and CD166,and in primitive CD34(+) CD38(-) and CD34(+) CD133(+) progenitors. Most of the CD34(+) and CD133(+) cells were ALDH(dim). ALDH(br) populations had 144-fold more hematopoietic colony-forming activity than ALDH(dim) cells and included all megakaryocyte progenitors. ALDH(br) populations readily established endothelial cell monolayers in cultures. Cells generating endothelial colonies in 7 days were 435-fold more frequent in ALDH(br) than ALDH(dim) populations. CFU-F were 9.5-fold more frequent in ALDH(br) than ALDH(dim) cells,and ALDH(br) cells gave rise to multipotential mesenchymal cell cultures that could be driven to develop into adipocytes,osteoblasts and chondrocytes. DISCUSSION: Hematopoietic,endothelial and mesenchymal progenitor cells can be isolated simultaneously from human BM by cell sorting based on ALDH activity. BM ALDH(br) populations may be useful in several cell therapy applications. View Publication

Ubiquitously acting chromatin opening elements (UCOEs) consist of methylation-free CpG islands encompassing dual divergently transcribed promoters of housekeeping genes that have been shown to confer resistance to transcriptional silencing and to produce consistent and stable transgene expression in tissue culture systems. To develop improved strategies for hematopoietic cell gene therapy,we have assessed the potential of the novel human HNRPA2B1-CBX3 UCOE (A2UCOE) within the context of a self-inactivating (SIN) lentiviral vector. Unlike viral promoters,the enhancer-less A2UCOE gave rise to populations of cells that expressed a reporter transgene at a highly reproducible level. The efficiency of expression per vector genome was also markedly increased in vivo compared with vectors incorporating either spleen focus-forming virus (SFFV) or cytomegalovirus (CMV) promoters,suggesting a relative resistance to silencing. Furthermore,an A2UCOE-IL2RG vector fully restored the IL-2 signaling pathway within IL2RG-deficient human cells in vitro and successfully rescued the X-linked severe combined immunodeficiency (SCID-X1) phenotype in a mouse model of this disease. These data indicate that the A2UCOE displays highly reliable transcriptional activity within a lentiviral vector,largely overcoming insertion-site position effects and giving rise to therapeutically relevant levels of gene expression. These properties are achieved in the absence of classic enhancer activity and therefore may confer a high safety profile. View Publication

Phosphoinositide 3-kinase (PI3K)-dependent signalling regulates a wide variety of cellular functions including proliferation and differentiation. Disruption of class I(A) PI3K isoforms has implicated PI3K-mediated signalling in development of the early embryo and lymphohaemopoietic system. We have used embryonic stem (ES) cells as an in vitro model to study the involvement of PI3K-dependent signalling during early development and haemopoiesis. Both pharmacological inhibition and genetic manipulation of PI3K-dependent signalling demonstrate that PI3K-mediated signals,most likely via 3-phosphoinositide-dependent protein kinase 1 (PDK1),are required for proliferation of cells within developing embryoid bodies (EBs). Surprisingly,the haemopoietic potential of EB-derived cells was not blocked upon PI3K inhibition but rather enhanced,correlating with modest increases in expression of haemopoietic marker genes. By contrast,PDK1-deficient EB-derived progeny failed to generate terminally differentiated haemopoietic lineages. This deficiency appeared to be due to a requirement for PI3K signalling during the proliferative phase of blast-colony-forming cell (BL-CFC) expansion,rather than as a result of effects on differentiation per se. We also demonstrate that PI3K-dependent signalling is required for optimal generation of erythroid and myeloid progenitors and their differentiation into mature haemopoietic colony types. These data demonstrate that PI3K-dependent signals play important roles at different stages of haemopoietic development. View Publication

Infection with a recombinant murine-feline gammaretrovirus,MoFe2,or with the parent virus,Moloney murine leukemia virus,caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective,in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state. View Publication

Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however,their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin(-))/CD45(+) BM cells,we found CD41(+) MKs without AChE activity (AChE(-)) except for CD41(++) MKs with AChE activity (AChE(+)),in which CD61 expression was similar to their CD41 level. Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs could differentiate into AChE(+),with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs were observed later than for Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs,whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions,including beta1-tubulin. In normal mice,the number of Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs was 100 times higher than that of AChE(+) MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent,mitomycin-C,Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs led to an increase in AChE(+) MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs and more differentiated Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs. Immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are a major MK population compared with AChE(+) MKs in BM and play an important role in rapid PLT recovery in vivo. View Publication

Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing,but its physiological role in vivo remains undefined. Here,we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews(-/-) lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre-B lymphocyte) development. During meiosis,Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination,resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence,and the mutant animals showed hypersensitivity to ionizing radiation. Finally,we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre-B cell development and meiosis,with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore,we demonstrate a novel role of EWS in cellular senescence,possibly through its interaction and modulation of lamin A/C. View Publication

Mesenchymal stem cells (MSC) are part of the bone marrow that provides signals supporting survival and growth of bystander hematopoietic stem cells (HSC). MSC modulate also the immune response,as they inhibit proliferation of lymphocytes. In order to investigate whether MSC can support survival of T cells,we investigated MSC capacity of rescuing T lymphocytes from cell death induced by different mechanisms. We observed that MSC prolong survival of unstimulated T cells and apoptosis-prone thymocytes cultured under starving conditions. MSC rescued T cells from activation induced cell death (AICD) by downregulation of Fas receptor and Fas ligand on T cell surface and inhibition of endogenous proteases involved in cell death. MSC dampened also Fas receptor mediated apoptosis of CD95 expressing Jurkat leukemic T cells. In contrast,rescue from AICD was not associated with a significant change of Bcl-2,an inhibitor of apoptosis induced by cell stress. Accordingly,MSC exhibited a minimal capacity of rescuing Jurkat cells from chemically induced apoptosis,a process disrupting the mitochondrial membrane potential regulated by Bcl-2. These results suggest that MSC interfere with the Fas receptor regulated process of programmed cell death. Overall,MSC can inhibit proliferation of activated T cells while supporting their survival in a quiescent state,providing a model of their activity inside the HSC niche. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer,including acute myeloid leukemia (AML),and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML,we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins,including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells,and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis,and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles. View Publication