Regulation of hematopoietic stem cell self-renewal by 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，因为这一知识可以应用于扩大临床有用的HSC数量用于骨髓移植和血液疾病的靶向基因治疗。许多研究表明，属于Hox基因家族的同源结构域蛋白在调节成人HSC自我更新中起重要作用。利用逆转录病毒载体在HSC中过表达同源盒基因Hoxb4，结果在体外长期再生的HSC （LT-HSC）数量增加40倍。当Hoxb6或Hoxa9在LT-HSC中过表达时，也观察到类似的LT-HSC对称自我更新的体外刺激。哺乳动物三胸同源物Mll的缺失导致体内成年LT-HSC的缺失，这进一步支持了Hox基因活性对HSC自我更新至关重要的假设。最近用逆转录病毒载体在小鼠造血祖细胞中表达急性髓系白血病癌蛋白NUP98-HOXA10的实验显示，在两周的体外培养期间，LT-HSC扩增了前所未有的10,000倍。在本提案中概述的研究中，我们观察到使用相关的Hox融合蛋白NUP98-HOXA9，成人LT-HSC在体外扩增了10,000倍。本研究的主要目的是利用遗传和生化方法描述NUP98- HOXA9介导的LT-HSC对称自我更新显著增强的分子机制。在Aim 1中，我们将从功能上测试NUP98-HOXA9、Hoxb4和Hoxa9在LT-HSC中普遍上调的基因是否有助于NUP98-HOXA9介导的对称自我更新。在Aim 2中，我们将描述Hoxa5和Hoxb5在多大程度上促进了NUP98-HOXA9刺激的强效HSC自我更新表型。在表达NUP98-HOXA9的LT-HSC中，Hoxa5和Hoxb5是两个上调最多的Hox基因，因此这两个同源基因可能在刺激LT-HSC扩增中具有独特的功能。最后，在Aim 3中，我们将定义Hox辅助因子Pbx1和Pbx3在由Hoxb4和NUP98-HOXA9介导的LT-HSC自我更新中的作用，并使用质谱法鉴定LT-HSC中NUP98-HOXA9相互作用蛋白。我们使用NUP98-HOXA9在体外将LT-HSC扩增10,000倍的能力将首次允许使用纯LT-HSC群体进行常规生物化学。这将有助于在LT-HSC中调节自我更新过程的Hox蛋白复合物的生化表征。总的来说，这些研究将为控制由Hox基因活性刺激的成人LT-HSC对称自我更新的基因和途径提供新的见解。

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.