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方案中，以扩大骨髓移植和血液病靶向基因治疗的临床有用数量的HSC。许多研究表明，Hox基因家族中含有同源结构域的蛋白质在调节成体HSC自我更新中起着重要作用。在HSC中使用逆转录病毒载体过表达同源异型盒基因Hoxb 4的实验导致体外长期再增殖HSC（LT-HSC）数量扩增40倍。当Hoxb 6或Hoxa 9在LT-HSC中过表达时，观察到类似的LT-HSC体外对称自我更新刺激。MII（一种哺乳动物三胸同源物，其功能是维持Hox基因表达）的丧失导致体内成年LT-HSC的丧失，这进一步支持了Hox基因活性对于HSC自我更新是必需的假设。最近的实验中，急性髓性白血病癌蛋白NUP 98-HOXA 10在小鼠造血祖细胞中使用逆转录病毒载体表达，显示出LT-HSC在两周的体外培养期内前所未有的10，000倍扩增。在本提案中概述的研究中，我们已经观察到使用相关Hox融合蛋白NUP 98-HOXA 9的成人LT-HSC的类似10，000倍体外扩增。该提案的主要目的是使用遗传和生物化学方法描绘负责NUP 98-HOXA 9介导的LT-HSC对称自我更新的显著增强的分子机制。在目的1中，我们将功能性地测试在LT-HSC中通常由NUP 98-HOXA 9、Hoxb 4和Hoxa 9上调的基因是否有助于NUP 98-HOXA 9介导的对称自我更新。对于目标2，我们将表征Hoxa 5和Hoxb 5对NUP 98-H 0XA 9刺激的有效HSC自我更新表型的贡献程度。Hoxa 5和Hoxb 5是表达NUP 98-HOXA 9的LT-HSC中上调最高的两个Hox基因，因此这些旁系同源基因可能在刺激LT-HSC扩增中具有独特的功能。最后，在目标3中，我们将定义Hox辅因子Pbx 1和Pbx 3在Hoxb 4和NUP 98-HOXA 9介导的LT-HSC自我更新中的作用，并使用质谱法鉴定LT-HSC中NUP 98-HOXA 9相互作用的蛋白。我们使用NUP 98-HOXA 9体外扩增LT-HSC 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Hox蛋白复合物的生物化学表征，这些蛋白复合物在LT-HSC中起作用以调节自我更新过程。总的来说，这些研究将为控制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。 公共卫生相关性：在体外扩增造血干细胞（HSC）至临床有用数量的能力在骨髓移植和血液病的基因治疗领域具有巨大的应用。在这个提议中，我们将探索由Hox融合蛋白NUP 98-HOXA 9刺激的成人骨髓HSC体外扩增10，000倍的潜在分子基础。

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.