Stem cell manipulation via HOX gene regulation

描述（申请人提供）：干细胞治疗在癌症治疗中发挥着重要且日益重要的作用。扩大干细胞数量的能力将在血液学和肿瘤学中有广泛的应用。此外，加强干细胞分化的能力可以改善许多癌症的治疗，特别是急性白血病。许多系统的生化和遗传实验表明，在许多组织中，同源盒蛋白调节干细胞和谱系特异性细胞之间的平衡。我们最近发现，三聚体转录因子NF-Y是调控HOXB4及其同类HOXC4和HOXD4表达的关键调控转录因子，而NF-Y此前已被证实可调控c-jun、p27和CD34的转录。通过逆转录病毒基因转移，三聚体的诱导因子NF-Ya在干细胞中的过度表达增加了HOXB4、HOXC4、HOXD4以及hTERT、LEF-1和几种干细胞标记物的表达。通过干细胞移植后的竞争性再生分析，干细胞中NF-Ya过表达也会增加干细胞数量，而抑制NF-Y活性会减少干细胞数量并促进终末分化。基于这些数据，我们假设NF-Y作为一个主开关，控制几个对干细胞循环和增殖至关重要的基因的表达，包括同源盒和其他基因。为了探索和验证这一假设，我们建议：1)在正常小鼠和几种候选下游NF-Y靶基因缺失的小鼠品系中，测量造血干细胞中NF-Ya过表达或过表达的后果；2)通过体内移植检测可溶性TAT-NF-Ya蛋白生化可逆激活NF-Y靶基因和增加造血干细胞的能力；3)体外检测DN-NF-Ya和TAT-DN- NF-Ya蛋白对原代AML母细胞的分化能力，以及体内对NOD/SCID人AML小鼠的分化能力。这些实验将详细描述NF-Y在造血干细胞生物学中的作用，并将开发直接基于NF-Y生物学的两种实验性疗法，用于HSC扩增和HSC分化。

DESCRIPTION (provided by applicant): Stem cell therapies play a major and increasing role in cancer therapies. The ability to expand stem cell numbers would have broad application in hematology and oncology. In addition, the ability to enforce stem cell differentiation could improve the therapy of many cancers, notably acute leukemias. Biochemical and genetic experiments in many systems indicate that homeobox proteins regulate the balance between stem cells and lineage-specific cells, in many tissues. We have recently found that the trimeric transcription factor NF-Y, which had been previously shown to regulate c-jun, p27 and CD34 transcription, is the key regulated transcription factor controlling the expression of HOXB4, as well as the paralogs HOXC4 and HOXD4. Enforced overexpression of NF-Ya, the inducible element of the trimer, in stem cells by retroviral gene transfer increases the expression of HOXB4, HOXC4, HOXD4, as well as hTERT, LEF-1 and several stem cell markers. NF-Ya overexpression in stem cells also increases stem cell numbers as assayed by competitive repopulation following stem cell transplantation, while inhibition of NF-Y activity decreases stem cell numbers and promotes terminal differentiation. Based upon these data, we hypothesize that NF-Y functions as a master switch, controlling the expression of several genes critical for stem cell cycling and proliferation, including homeobox and other genes. To explore and test this hypothesis, we propose to: 1) Measure the consequences of NF-Ya over/ and underexpression in hematopoietic stem cells, both in normal mice and mouse strains deficient in each of several candidate downstream NF-Y target genes; 2) test the ability of soluble TAT-NF-Ya protein to biochemically and reversibly activate NF-Y target genes and increase HSCs, as measured by transplantation in vivo; and 3) Test the ability of DN-NF-Ya, and TAT-DN- NF-Ya protein to differentiate primary AML blasts in vitro, and on NOD/SCID mice with human AML in vivo. These experiments will describe in detail the role of NF-Y in the biology of hematopoietic stem cells, and will develop two experimental therapies based directly on the biology of NF-Y, for both HSC expansion and HSC differentiation.