Wnt Signaling in Hematopoietic Stem Cell Specification and Leukemia

造血干细胞规格和白血病中的 Wnt 信号转导

• 批准号: 8535188
• 负责人: WILSON Kendrick Clements
• 金额: $ 23.12万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535188
• 关键词: Ablation; Acute; Acute Lymphocytic Leukemia; Acute leukemia; Alleles; Animal Model; Animals; Antineoplastic Agents; Aorta; B-Cell Acute Lymphoblastic Leukemia; B-Lymphocytes; Behavior; Biology; Blood; Blood Cells; Caenorhabditis elegans; Cell Transplantation; Cells; Chronic; Chronic Lymphocytic Leukemia; Commit; Data; Defect; Development; Disease; Disease Progression; Dominant-Negative Mutation; Dorsal; Embryo; Embryonic Development; Endothelium; Erythrocytes; Event; Failure; Family; Family member; Future; Gene Expression; Gene Transfer Techniques; Genes; Genetic; Goals; Hematopoietic; Hematopoietic stem cells; Imagery; Immune; Individual; Investigation; Label; Lesion; Ligands; Lymphoblastic Leukemia; Modeling; Mutation; Natural regeneration; Nature; Oncogenes; Orthologous Gene; Pathway interactions; Phenotype; Process; Production; Proteins; RORA gene; Regenerative Medicine; Regulation; Research; Sclerotome; Signal Transduction; Signal Transduction Pathway; Smooth Muscle Myocytes; Somites; Specific qualifier value; Staging; Stem cells; TCF3 gene; Testing; Time; Tissues; To specify; Transcriptional Activation; Transgenic Animals; Transgenic Organisms; Vertebrates; Zebrafish; abstracting; cancer initiation; cell type; clinically relevant; combinatorial; congenital blood disorder; drug discovery; embryonic stem cell; hematopoietic stem cell fate; induced pluripotent stem cell; leukemia; leukemogenesis; member; mutant; notch protein; novel; primitive cell; receptor; research study; self-renewal; stem cell fate specification; tissue culture

Project Summary/Abstract Hematopoietic stem cells (HSCs) are progenitor cells that have the ability to both self-renew and regenerate all mature blood cell types, including red blood cells and immune cells over the lifetime of an individual. HSCs are used therapeutically in the treatment of numerous diseases including leukemia and congenital blood disorders, but obtaining suitable numbers of histocompatible cells for transplantation remains a problem. Determining how embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) are directed to become tissue specific stem cells such HSCs is a key goal of regenerative medicine. The most obvious approach to defining required regulatory networks is to determine the endogenous mechanisms used during embryonic development. Often, involved signal transduction pathways are also observed to be dysregulated in leukemia, making an understanding of their basic biology of further clinical relevance. I have recently shown that "non- canonical", ¿-catenin/Tcf-independent signaling by the Wnt pathway, which was not previously known to be involved in HSC specification, is required for formation HSCs. The Wnt ligand, Wnt16, which is conserved across vertebrate phyla and was originally identified as a gene aberrantly upregulated in pre-B acute lymphocytic leukemia (ALL), is required for specification of the first HSCs. My preliminary results show that Wnt16 activates expression of two Notch ligands, deltaC and deltaD, and that these ligands are in turn required redundantly for HSC specification. Although cell-autonomous reception of a Notch signal in cells fated to become HSCs is an established requirement in vertebrates, the Notch signaling events regulated by DeltaC and DeltaD appear to be non-cell-autonomous and therefore represent a distinct, previously unappreciated requirement for Notch signaling. In the research proposed here, I will seek to determine the precise signal transduction pathway(s) that lie between Wnt16 and transcriptional activation of deltaC and deltaD, by identifying the required co-receptors and intracellular signal transduction proteins. Preliminary data suggest that absence of DeltaC and DeltaD leads to defects in the formation or behavior of a somite compartment, the sclerotome, which is adjacent to the primitive dorsal aorta, the tissue that gives rise to the first HSCs in vertebrates. During the time when endothelium of the dorsal aorta becomes hemogenic and commits to an HSC fate, sclerotomally derived cells emigrate from the somite to become smooth muscle cells surrounding the aorta and may also directly contribute "replacement" endothelium, suggesting that sclerotomal defects underlie failure of HSC specification. Using zebrafish, which are transparent during embryonic development and uniquely receptive to transgenesis, allowing direct visualization of fluorescently labeled tissues, I will generate transgenic animals with labeled sclerotome, to determine how this tissue behaves in normal and Wnt16/Notch-deficient animals. I will test the overall requirement for sclerotome in development by conditional ablation, and the requirement for specific proteins in sclerotomal cells by conditional expression of wild-type and dominant negative factors. The non-canonical Wnt receptor, Ryk appears to participate in Wnt16 signal transduction, but cannot explain all of the effects observed in Wnt16 deficient animals. These results suggest the presence of additional co-receptors. The strongest family of candidates for this co-receptor is the Ror family of non- canonical Wnt receptors. The C. elegans ortholog of Wnt16, EGL-20 interacts physically and functionally with the single worm Ror ortholog, CAM-1. Zebrafish have three Ror family members, MuSK, Ror1, and Ror2. I have determined that Ror2 is not the required Wnt16 co-receptor, and the phenotype of the unplugged mutant, which carries a null allele of the musk gene, suggests that MuSK is also unlikely to contribute to the Wnt16 hematopoietic phenotype. Thus, Ror1 is the strongest candidate for the Wnt16 co-receptor. Interestingly, ROR1 misexpression is strongly associated with chronic lympocytic leukemia (CLL) as well as some ALL. WNT16 is also misexpressed in forms of ALL and CLL. Taken together with the fact that tissue culture experiments suggest that WNT16 is causally involved in pre-B-ALL, these results suggest that WNT16 and ROR1 may cooperatively or independently contribute to leukemogenesis. To test these possibilities, I will generate transgenic animals in which wnt16, ror1, and the oncogene E2A-PBX1, which has previously been associated with WNT16-directed disease progression in pre-B-ALL, are expressed in B-cells at a variety of maturation stages. Since no initiating lesions are known for CLL, these models have the potential to be extremely informative. Finally, I will use the B-cell transgenic animals as a platform for unbiased discovery of additional mutations that are involved in B-cell leukemia by forward genetics.

项目总结/文摘