Stem cell manipulation via HOX gene regulation

描述(由申请人提供)：干细胞疗法在癌症治疗中发挥着越来越重要的作用。扩大干细胞数量的能力将在血液学和肿瘤学中得到广泛应用。此外，促进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。在许多系统中的生化和遗传学实验表明，在许多组织中，同源框蛋白调节干细胞和特定血统细胞之间的平衡。我们最近发现，三聚体转录因子NF-Y是控制HOXB4以及同源基因HOXC4和HOXD4表达的关键转录因子，此前已发现它调控c-jun、p27和CD34的转录。通过逆转录病毒基因转移在干细胞中强制过表达三聚体的诱导元件NF-Ya会增加HOXB4、HOXC4、HOXD4以及hTERT、Lef-1和几个干细胞标记物的表达。根据干细胞移植后竞争性再繁殖的分析，干细胞中过表达核因子-Ya也增加了干细胞数量，而抑制核因子-Y活性则减少了干细胞数量并促进了终末分化。基于这些数据，我们假设核因子-Y作为主开关发挥作用，控制几个对干细胞周期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说，我们建议：1)测量NF-Ya在正常小鼠和缺少几个候选下游NF-Y靶基因的小鼠品系中高/低表达的后果；2)测试可溶性TAT-NF-Ya蛋白在生物化学上和体内移植后可逆地激活NF-Y靶基因和增加HSCs的能力；3)测试dN-NF-Ya和TAT-dN-NF-Ya蛋白在体外以及在NOD/SCID小鼠和人AML小鼠身上分化原代AML细胞的能力。这些实验将详细描述核因子-Y在造血干细胞生物学中的作用，并将直接基于核因子-Y的生物学开发两种实验性疗法，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.