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: 8111654
• 负责人: WILSON Kendrick Clements
• 金额: $ 13.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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 2012-09-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8111654
• 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; Health; 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; 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

DESCRIPTION (provided by applicant): 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", 2-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.

描述（由申请人提供）：造血干细胞（hsc）是一种祖细胞，具有自我更新和再生所有成熟血细胞类型的能力，包括个体一生中的红细胞和免疫细胞。造血干细胞被用于治疗包括白血病和先天性血液疾病在内的许多疾病，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）如何定向成为组织特异性干细胞（如hsc）是再生医学的关键目标。定义所需调控网络的最明显方法是确定胚胎发育过程中使用的内源性机制。通常，相关的信号转导通路在白血病中也被观察到失调，这使得对其基本生物学的理解具有进一步的临床意义。我最近的研究表明，“非规范的”、2-catenin/ tcf独立的Wnt通路信号传导是形成HSC所必需的，而这在以前并不被认为与HSC规范有关。Wnt配体Wnt16在整个脊椎动物门中都是保守的，最初被鉴定为在b前急性淋巴细胞白血病（ALL）中异常上调的基因，是第一批造血干细胞特异性所必需的。我的初步结果表明，Wnt16激活了两个Notch配体，deltaC和deltaD的表达，而这些配体对于HSC规范又是冗余的。虽然在脊椎动物中注定成为造血干细胞的细胞中，Notch信号的细胞自主接收是一种既定的需求，但由DeltaC和DeltaD调节的Notch信号事件似乎是非细胞自主的，因此代表了一种不同的、以前未被认识到的Notch信号需求。在这里提出的研究中，我将通过鉴定所需的共受体和细胞内信号转导蛋白，寻求确定Wnt16与deltaC和deltaD转录激活之间的精确信号转导途径。初步数据表明，DeltaC和DeltaD的缺失会导致体隔室（靠近原始背主动脉的硬膜组）的形成或行为缺陷，而硬膜组是脊椎动物中产生第一批造血干细胞的组织。当背主动脉内皮成为造血细胞并形成HSC时，硬化层来源的细胞从小体迁移成为主动脉周围的平滑肌细胞，并可能直接提供“替代”内皮，提示硬化层缺陷是HSC规范失败的基础。利用斑马鱼，在胚胎发育过程中是透明的，并且具有独特的转基因接受能力，可以直接看到荧光标记的组织，我将产生带有标记核组的转基因动物，以确定该组织在正常和Wnt16/缺口缺陷动物中的行为。我将通过条件性消融术来测试发育过程中对硬膜组的总体需求，通过野生型和显性负性因子的条件性表达来测试硬膜细胞对特定蛋白质的需求。非典型Wnt受体Ryk似乎参与Wnt16信号转导，但不能解释在Wnt16缺陷动物中观察到的所有影响。这些结果表明存在额外的共受体。这种共受体的最强候选家族是Ror家族的非典型Wnt受体。线虫Wnt16的同源物EGL-20在物理和功能上与单个线虫Ror同源物CAM-1相互作用。斑马鱼有三个Ror家族成员，麝香，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（携带麝香基因的空等位基因）表明，麝香也不太可能促成Wnt16造血表型。因此，Ror1是Wnt16共受体的最强候选。有趣的是，ROR1错误表达与慢性淋巴细胞白血病（CLL）以及一些ALL密切相关。WNT16在ALL和CLL中也存在错误表达。结合组织培养实验表明WNT16与b - all前期有因果关系的事实，这些结果表明WNT16和ROR1可能协同或独立地促进白血病的发生。为了测试这些可能性，我将产生转基因动物，其中wnt16， ror1和致癌基因E2A-PBX1在不同成熟阶段的b细胞中表达，这些基因先前与wnt16指导的b - all前期疾病进展有关。由于CLL没有已知的初始病变，因此这些模型有可能提供非常丰富的信息。最后，我将使用b细胞转基因动物作为平台，通过正向遗传学公正地发现与b细胞白血病有关的其他突变。