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  • CHEMISTRY; METALLURGY
    • BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
      • MEASURING OR TESTING PROCESSES INVOLVING ENZYMES OR MICRO-ORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
      • MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF (biocides, pest repellants or attractants, or plant growth regulators, containing micro-organisms, viruses, microbial fungi, enzymes, fermentates or substances produced by or extracted from micro-organisms or animal material A01N63/00; food compositions A21, A23; medicinal preparations A61K; chemical aspects of, or use of materials for, bandages, dressings, absorbent pads or surgical articles A61L; fertilisers C05); PROPAGATING, PRESERVING OR MAINTAINING MICRO-ORGANISMS (preservation of living parts of humans or animals A01N1/02); MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (micro-biological testing media C12Q)
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Bibliografia
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Keywords
LEUKEMIC STEM CELLS, ACUTE MYELOBLASTIC LEUKEMIA, P2 RECEPTORS, GENE EXPRESSION PROFILE, GENE SILENCING, XENOGENIC TRANSPLANT, DIFFERENTIATING AGENTS, EXTRACELLULAR NUCLEOTIDES

Isolation, molecular and functional characterization of leukemic stem cells (LSCs) for the development of novel strategies of target therapy

Università degli Studi di Bologna
Abstract
· BACKGROUND There is increasing evidence that the leukemic clone is sustained by a rare population of leukemic stem cells (LSCs) that have acquired the ability of self-renewing and are capable of transferring acute myeloblastic leukemia (AML) from human patients to immuno compromised mice. Isolation, molecular and functional characterization of the myeloid cell undergoing leukemic transformation and the resultant LSCs that initiate and maintain AML is essential for both the understanding of the leukemogenic process and for the design of effective therapies targeting specific molecular defects of AML.
· OBJECTIVES The project aims to the 1) isolation and functional characterization, in vitro and in vivo, of LSCs; 2) identification of the moleculare signature of LSCs by microarray technology; 3)induction of leukemic phenotype by transduction into normal hematopoietic stem cells (HSC) of leukemia-related genes by lentiviral vectors; 4) silencing of leukemia-related genes in LSCs; 5) investigation on the responsiveness of LSCs to novel anti-proliferative and differentiating agents designed on the molecular differences between normal and leukemic hematopoiesis (targeted therapy).
· PROJECT The project is divided in 4 Tasks. In Task 1, LSCs will be purified by immunomagnetic and cell sorting technology as CD38-Lin-CD34+ CD123+or CD38-Lin-CD34+CD90- cells. LSCs and unselected AML blasts will be characterized by flow cytometric evaluation of the cell cycle >>>

Principal Investigator
Roberto Massimo Lemoli Università degli Studi di BOLOGNA
Research Objectives
Blast cells of acute myeloblastic leukemia (AML) have a limited proliferative capacity. There is increasing evidence that the leukemic clone is sustained by a rare population of leukemic stem cells (LSCs) that have acquired the ability of self-renewing. Elucidating the nature of the myeloid cell undergoing leukemic transformation and the resultant LSCs that initiate and maintain AML is essential for both the understanding of the leukemogenic process and for the design of effective therapies targeting specific molecular defects of AML. In this view, recent studies have demonstrated that new subgroups of AML with characteristic gene-expression signatures, and different prognosis, can be identified with the microarray technology. Thus, the application of genome-wide arrays allows the analysis of minimal differences in gene expression between normal and leukemic myeloid cells. However, these investigations have been conducted on bulk populations of AML cells and not on LSCs which are crucial for the development of the neoplasia. Identification and isolation of LSCs would facilitate the study of gene expression profiles of malignant progenitors compared with their normal counterparts by using microarray technology. The identification of genes expressed in LSCs should therefore lead to novel therapies directed toward gene products expressed in LSCs but not in hematopoietic stem cells (HSCs). Purified LSCs will also provide a powerful substrate for preclinical testing of the >>>

Timescale
24 months
National and international background
Emerging evidence indicates that AML derives from LSCs that can either be hematopoietic stem cells (HSCs) that have become leukemic as the result of accumulated mutations or more restricted progenitors that have reacquired the stem cell capability for self-renewal. Most of the leukemic cells are unable to proliferate extensively and only a small and phenotypically defined subset of cells (i.e. CD34+CD38-) is clonogenic and capable of transferring AML from humans to nonobese diabetic/severe combined immunedeficient (NOD/SCID) mice (1). Conversely, except for the M3 acute promyelocytic leukemia (APL) subtype, more mature CD34+CD38+ progenitors can not transfer the disease to NOD/SCID mice. These observations suggest that for most AML subtypes HSCs rather than committed progenitors are the target for leukemic transformation regardless of the lineage of differentiation of the leukemic cells circulating into peripheral blood. In APL, the transformation process may involve a more differentiated hematopoietic cell as demonstrated by the expression of the t(15; 17) balanced reciprocal translocation and the M3-associated fusion gene PML/RAR-alfa in CD34-CD38+ cells but not in CD34+CD38- HSCs (2). Although there is strong evidence for the origin of most AML at the HSCs level, further definition of the phenotype of LSCs has revealed some differences between leukemic and normal stem cells. While LSCs appear to share many of the cell surface markers previously identified for HSCs such as >>>