Regulation of hematopoietic stem cell self-renewal by NUP98-HOXA9

NUP98-HOXA9对造血干细胞自我更新的调控

基本信息

批准号：
8006300
负责人：
CHRISTOPHER KLUG
金额：
$ 36.63万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-29 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8006300
关键词：
Acute Myelocytic Leukemia Adult Area Biochemical Biochemistry Bone Marrow Bone Marrow Transplantation Cell Aging Cell Count Cell Cycle Cell Maintenance Cell division Chimeric Proteins Chromatin Complex Disease Fetal Liver Gene Expression Gene Family Genes Genetic Goals HOXA9 gene Hematopoietic stem cells Homeobox Genes Homeostasis Homologous Gene Human In Vitro Knowledge Maintenance Mass Spectrum Analysis Mediating Molecular Mus NUP98 gene Names Oncogene Proteins Pathway interactions Phenotype Phosphoric Monoester Hydrolases Population Process Proteins Protocols documentation Regulation Research Retroviral Vector Role Signal Transduction Signaling Molecule Stem cells Testing Time Transcript Transplantation base gene therapy homeodomain in vivo insight loss of function overexpression paralogous gene protein complex public health relevance research study self renewing cell self-renewal stem cell niche trafficking transcription factor

项目摘要

DESCRIPTION (provided by applicant): One of the most elusive goals in hematologic research has been to understand how hematopoietic stem cells (HSC) self-renew, since this knowledge could be applied in protocols to expand clinically useful numbers of HSC for bone marrow transplantation and targeted gene therapy for hematologic disorders. A number of studies have suggested an essential role for homeodomain-containing proteins belonging to the Hox gene family in the regulation of adult HSC self-renewal. Experiments where the homeobox gene, Hoxb4, was overexpressed using a retroviral vector in HSC resulted in a 40-fold expansion in long-term repopulating HSC (LT-HSC) numbers in vitro. A similar stimulation of LT-HSC symmetric self-renewal in vitro was observed when Hoxb6 or Hoxa9 were overexpressed in LT-HSC. Loss of Mll, a mammalian trithorax homolog that functions to maintain Hox gene expression, results in the loss of adult LT-HSC in vivo, which further supports the hypothesis that Hox gene activity is essential for HSC self-renewal. Recent experiments where the acute myeloid leukemia oncoprotein NUP98-HOXA10 was expressed in murine hematopoietic progenitor cells using a retroviral vector showed an unprecedented 10,000-fold expansion of LT-HSC over a two-week in vitro culture period. In studies outlined in this proposal, we have observed a similar 10,000-fold in vitro expansion of adult LT-HSC using a related Hox fusion protein, NUP98-HOXA9. The major objective of this proposal will be to delineate the molecular mechanisms responsible for the dramatic enhancement of LT-HSC symmetric self-renewal mediated by NUP98- HOXA9 using genetic and biochemical approaches. In Aim 1, we will functionally test whether genes commonly up-regulated in LT-HSC by NUP98-HOXA9, Hoxb4, and Hoxa9 contribute to NUP98-HOXA9- mediated symmetric self-renewal. For Aim 2, we will characterize the extent to which Hoxa5 and Hoxb5 contribute to the potent HSC self-renewal phenotype stimulated by NUP98-HOXA9. Hoxa5 and Hoxb5 are the two most highly up-regulated Hox genes in LT-HSC expressing NUP98-HOXA9 so these paralogous genes may have a unique function in stimulation of LT-HSC expansion. Finally, in Aim 3 we will define the roles of the Hox co-factors, Pbx1 and Pbx3, in LT-HSC self-renewal mediated by Hoxb4 and NUP98- HOXA9 and identify NUP98-HOXA9-interacting proteins in LT-HSC using mass spectrometry. Our ability to expand LT-HSC 10,000-fold in vitro using NUP98-HOXA9 will allow, for the first time, conventional biochemistry to be done using a pure population of LT-HSC. This will facilitate the biochemical characterization of Hox protein complexes that are functioning in LT-HSC to regulate the self-renewal process. Collectively, these studies will provide new insights into the genes and pathways controlling adult LT-HSC symmetric self-renewal that is stimulated by Hox gene activity. PUBLIC HEALTH RELEVANCE: The ability to expand hematopoietic stem cells (HSC) in vitro to clinically useful numbers has tremendous application in the areas of bone marrow transplantation and gene therapy for hematologic disorders. In this proposal, we will explore the underlying molecular basis for the dramatic 10,000-fold ex vivo expansion of adult bone marrow HSC that is stimulated by the Hox fusion protein, NUP98-HOXA9.

描述（由申请人提供）：血液学研究中最难以捉摸的目标之一是了解造血干细胞（HSC）如何自我更新，因为可以将这些知识应用于方案中，以扩大临床上有用的HSC数量的HSC，用于骨髓移植和靶向血液学疾病的基因治疗。许多研究表明，在成人HSC自我更新的调节中，属于HOX基因家族的含同源域蛋白的重要作用。 HSC中使用逆转录病毒载体过表达同源基因HOXB4的实验，导致长期重新流传HSC（LT-HSC）数量的40倍膨胀。当LT-HSC中过表达HOXB6或HOXA9时，观察到LT-HSC对称自我更新的类似刺激。 MLL的丧失是一种哺乳动物的Trithorax同源物，可维持HOX基因表达，导致体内成年LT-HSC的丧失，这进一步支持了HOX基因活性对于HSC自我更新至关重要的假设。最近的实验，急性髓细胞性白血病NUP98-HOXA10在使用逆转录病毒载体的鼠造血祖细胞中表达了肌肉造血祖细胞，显示了在两周的体外培养时期内LT-HSC的前所未有的10,000倍。在该提案中概述的研究中，我们使用相关的HOX融合蛋白NUP98-HOXA9观察到了成人LT-HSC的10,000倍体外扩张。该提案的主要目的是描述负责使用遗传和生化方法介导的LT-HSC对称自我更新LT-HSC对称自我更新的分子机制。在AIM 1中，我们将在功能上测试NUP98-HOXA9，HOXB4和HOXA9在LT-HSC中通常上调的基因是否有助于NUP98-HOXA9-介导的对称自我恢复。对于AIM 2，我们将表征HOXA5和HOXB5对NUP98-HOXA9刺激的有效HSC自我更新表型的贡献的程度。 HOXA5和HOXB5是表达NUP98-HOXA9的LT-HSC中两个最高上调的HOX基因，因此这些副基因可能在刺激LT-HSC膨胀方面具有独特的功能。最后，在AIM 3中，我们将定义HOXB4和NUP98-HOXA9介导的LT-HSC自我更新中HOX辅助因子PBX1和PBX3的作用，并识别LT-HSC在LT-HSC中使用质谱法中的NUP98-Hoxa9-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa-Hoxa9。我们使用NUP98-HOXA9在体外扩展LT-HSC 10,000倍的能力将首次使用LT-HSC的纯种群进行常规的生物化学。这将促进在LT-HSC中起作用以调节自我更新过程的HOX蛋白复合物的生化表征。总的来说，这些研究将提供有关由HOX基因活性刺激的成年LT-HSC对称自我更新的基因和途径的新见解。公共卫生相关性：在体外扩展造血干细胞（HSC）到临床上有用的数字的能力在血液学疾病中具有巨大的应用。在此提案中，我们将探索由HOX融合蛋白NUP98-HOXA9刺激的成年骨髓HSC的10,000倍离体扩增的基本分子基础。