MLL in Hematopoiesis and Leukemia in the Zebrafish Model

描述(申请人提供)：婴儿白血病和化疗相关白血病的特点是染色体带11q23的MLL基因易位，是具有独特临床和生物学特征的毁灭性疾病，通常是致命的。MLL编码一种具有转录抑制和激活功能的复杂癌蛋白。易位涉及多个伙伴基因，导致5‘-MLL-Partner-3’重排、5‘-Partner-MLL-3’重排和MLL单倍体不足，因为易位涉及一个等位基因。斑马鱼胚胎的独特属性使其能够在体内可视化完整动物的正常和异常的造血发育过程，这是其他动物模型所不能比拟的。此外，斑马鱼胚胎非常适合建立MLL模型，因为婴儿白血病发生中的MLL易位起源于子宫。到目前为止，还没有对斑马鱼进行MLL的研究。我们克隆了斑马鱼MLL同源基因，并显示了人类MLL的所有关键功能域的高度保守性。我们发现MLL转录本是母体提供给胚胎的，在斑马鱼的整个生命周期中都有表达，在小鼠的造血细胞和其他表达MLL的组织中可以检测到。我们证明了MLL-/-小鼠的外周表型、细胞凋亡和贫血都与MLL-/-小鼠的表型、细胞凋亡和贫血有关。我们进行了引人注目的观察，结果表明MLL的缺失不仅导致同源盒辅因子减少和细胞周期基因表达改变，而且还导致许多造血基因在不同的血细胞室中表达过高和表达不足，这表明MLL与以前未知的靶点有新的联系。此外，血细胞基因表达的变化导致了深刻的血细胞畸形。虽然在前体/髓系中高表达的基因包括Lmo2、scl、cmyb、Ikaros、rag2和GATA1，但在红系部分检测到GATA1和其他红细胞基因的表达减少，将MLL耗竭的贫血与整个红系程序的放松调节联系在一起。这导致了一种假设，即MLL在造血系统的发育控制中具有深刻的多谱系作用，MLL对基因表达的时间和细胞类型的特异性调节包括抑制和激活是造血祖细胞和干细胞发育有序规范所必需的，MLL的丢失导致血细胞系基因的异位和异步过度表达和低表达，以及造血系统的无效发育。该项目致力于通过利用整个生物体研究以及对整个生物体细胞的细胞和分子研究来研究这一假说，这些研究只有在斑马鱼中才可能，以确定MLL最重要的细胞以及MLL易位第一次转化的时间。 公共卫生相关性：MLL是一种对血细胞发育至关重要的基因，其称为易位的异常对受白血病影响的婴儿人群和某些抗癌药物的并发症受白血病影响的患者人群的发病率和死亡率有重大影响。该项目将利用斑马鱼的巨大潜力，了解从活胚胎最早的发育时间点开始，MLL如何在空间和时间上调节正常血细胞发育的动力学，以及同样的动力学如何在疾病中解除调节。利用斑马鱼来模拟人类MLL在调节正常和恶性血细胞发育中的功能，将简化未来为MLL白血病患者提供新的分子靶向治疗的途径。

DESCRIPTION (provided by applicant): Infant leukemias and chemotherapy related leukemias characterized by translocations of the MLL gene at chromosome band 11q23 are devastating, often fatal diseases with unique clinical and biological features. MLL encodes a complex oncoprotein with transcriptional repression and activation functions. The translocations involve many partner genes and generate 5'-MLL-Partner-3' rearrangements, 5'-Partner-MLL-3' rearrangements and MLL haploinsufficiency due to involvement of one allele in the translocation. Unique attributes of zebrafish embryos enable in vivo visualization of normal and abnormal hematopoietic developmental processes in intact animals like no other animal models. Moreover, zebrafish embryos are well suited to model MLL because MLL translocations in infant leukemogenesis originate in utero. Until now, no studies of mll had been done in zebrafish. We cloned the zebrafish mll ortholog and showed high conservation of all of the critical functional domains of human MLL. We found that mll transcripts are maternally supplied to the embryo, expressed during the entire zebrafish lifespan, and detectable in hematopoietic cells and other tissues where Mll is expresed in mice. We demonstrated that mll morpholino knockdown phenocopies the external phenotype, apoptosis and anemia of Mll-/- mice. We made the striking observations that the resulting mll depletion caused not only reduced homeobox cofactor and altered cell cycle gene expression, but also overexpression and underexpression of many hematopoietic genes in different blood cell compartments, suggesting new links of mll to previously unknown targets. Furthermore, the changes in blood cell gene expression caused profound blood cell dysmorphologies. While overexpressed genes in the precursor/myeloid compartment featured lmo2, scl, cmyb, ikaros, rag2 and gata1, reduced expression of gata1 and other red cell genes was detected in the erythroid fraction, linking the anemia from mll depletion to deregulation of an entire erythroid program. This leads to the hypothesis that MLL has profound multi-lineage roles in the developmental control of the hematopoietic system, that temporal and cell-type specific regulation of gene expression by MLL including repression and activation is required for orderly specification of hematopoietic progenitor and stem cell development, and that loss of MLL causes ectopic and asynchronous overexpression and underexpression of blood cell lineage genes and ineffective development of the hematopoietic system. This project endeavors to investigate this hypothesis by exploiting whole organism studies and cellular and molecular studies on cells from whole organisms that are only possible in zebrafish to pinpoint the cells where MLL is most important and when MLL translocations first become transforming. PUBLIC HEALTH RELEVANCE: MLL is a critically important gene for blood cell development, abnormalities of which called translocations have a significant impact on morbidity and mortality in the infant population affected with leukemia and in the patient population affected with leukemia as a complication of certain anti-cancer drugs. This project will exploit the tremendous potential of zebrafish to understand the dynamics of how normal blood cell development is regulated by MLL in space and time from the earliest developmental timepoints in the living embryo, and how the same dynamics are deregulated in disease. The exploitation of zebrafish to model the functions of human MLL in the regulation of normal and malignant blood cell development will streamline future inroads to new molecularly targeted therapies for patients with MLL leukemia.