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: 8529684
• 负责人: WILSON Kendrick Clements
• 金额: $ 24.9万
• 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/8529684
• 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.

项目摘要/摘要 造血干细胞(HSCs)是具有自我更新和再生ALL能力的祖细胞。 成熟血细胞类型，包括一个人一生中的红细胞和免疫细胞。HSC是 用于治疗包括白血病和先天性血液疾病在内的多种疾病， 但获得合适数量的组织相容细胞用于移植仍然是一个问题。确定 胚胎干细胞(ESCs)或诱导多能干细胞(IPSCs)是如何定向成为组织的 特定的干细胞，如造血干细胞，是再生医学的关键目标。最明显的方法是定义 所需的调控网络是确定胚胎期间使用的内源性机制 发展。通常，白血病中涉及的信号转导通路也被观察到是失调的， 对它们的基础生物学有进一步的临床意义。我最近展示了“非-- 典型的“，？-连环蛋白/Tcf-非依赖的信号通过Wnt途径，这是以前未知的 HSC规范中所涉及的，是组建HSC所必需的。保守的Wnt配体Wnt16 在脊椎动物门类中，最初被鉴定为在B型前急性加重期异常上调的基因 淋巴细胞白血病(ALL)是第一批造血干细胞的规范所必需的。我的初步结果显示 WNT16激活两个Notch配体，DeltaC和DeltaD的表达，并且这些配体依次 HSC规范需要冗余。尽管小区中的陷波信号的小区自主接收 注定要成为HSCs是脊椎动物的既定要求，Notch信号事件受 DeltaC和DeltaD似乎是非细胞自治的，因此代表了一个不同的，以前 对Notch信令的不受欢迎的要求。在这里提出的研究中，我将寻求确定 位于Wnt16和DeltaC转录激活之间的精确信号转导途径(S) DeltaD，通过鉴定所需的辅受体和细胞内信号转导蛋白。初步数据 提示缺少DeltaC和DeltaD会导致体节的形成或行为出现缺陷 隔室，与原始背主动脉相邻的硬化孔，该组织产生 首次在脊椎动物中发现了造血干细胞。在背主动脉内皮变得血液化和 致力于HSC的命运，硬核来源的细胞从体节迁徙成为平滑肌细胞 和可能直接贡献“替代”的内皮，提示硬化症 缺陷是HSC规范失败的根本原因。使用斑马鱼，它们在胚胎期间是透明的 开发和独特地接受转基因，允许直接显示荧光标记的 组织，我将产生带有标记菌核组织的转基因动物，以确定这种组织在 正常和Wnt16/Notch缺陷动物。我将通过以下方式测试开发中的skerotome的总体需求 条件消融，以及通过条件表达对硬化症细胞中特定蛋白质的需求 野生型和显性负面因素。 非规范的Wnt受体Ryk似乎参与了Wnt16的信号转导，但不能 解释在Wnt16缺陷动物身上观察到的所有影响。这些结果表明， 额外的辅助受体。这种共同受体的最强候选家族是Ror家族的非 典型的Wnt受体。线虫Wnt16、EGL-20的直系同源基因在物理和功能上与 单蜗杆罗尔直通仪，CAM-1。斑马鱼有三个Ror家族成员，穆斯克、Ror1和Ror2。我 已经确定Ror2不是所需的Wnt16共受体，并且未插入突变体的表型， 携带麝香基因的零等位基因，这表明麝香也不太可能对WNT16起作用 造血表型。因此，Ror1是Wnt16共同受体的最强候选者。有趣的是， ROR1的错误表达与慢性淋巴细胞白血病(CLL)以及某些ALL密切相关。 WNT16也以ALL和CLL的形式错误表达。结合组织培养这一事实 实验表明WNT16与前B-ALL有因果关系，这些结果提示WNT16和 ROR1可能协同或独立参与白血病的发生。为了测试这些可能性，我将 产生转基因动物，在其中wnt16、ROR1和癌基因E2a-Pbx1，以前已经被 与WNT16导向的疾病进展相关的前B-ALL，在B细胞中表达在不同的 成熟期。由于CLL尚无始发损害的已知，这些模型有可能是 信息量非常大。最后，我将利用B细胞转基因动物作为一个平台，公正地发现 通过正向遗传学与B细胞白血病有关的其他突变。