Ras-PI3K Pathway in Nf1-/-Hematopoiesis and Leukemia

描述(申请人提供)：神经纤维瘤病1型(NF1)是一种由NF1肿瘤抑制基因突变引起的遗传性疾病。NF1患儿易患幼年性粒单核细胞白血病(JMML)。神经纤维蛋白是NF1的蛋白产物，是p21ras活性的负调控因子。尽管NF1-/-小鼠在子宫中死亡，但用NF1-/-胎儿干细胞重组的小鼠患上了类似于NF1患者JMML的骨髓增生性疾病(MPD)。然而，导致MPD的NF1缺陷细胞中p21ras信号通路的变化尚不清楚。利用PI-3激酶(PI3K)抑制剂，我们有初步数据表明，p21ras-PI3K通路的过度激活与NF1-/-细胞的过度增殖和提高存活率有关。然而，使用PI3K抑制剂对结果的解释是有限的，因为有四类PI3K，而抑制剂使所有类别的PI3K失活。在这里，我们建议进行基因实验，以确定IAPI3K类的过度激活是否会改变NF1-/-造血细胞的生长。我们在NF1-/-细胞中学习IAPI3K类的理由有两个。首先，与其他PI3K不同，所有的IAPI3K催化亚基都含有一个p21ras结合域。第二，在体外，p21ras与这些亚基相互作用以增强激酶活性，并且没有证据表明p21ras增强其他PI3K类的活性。最近，一种p85pha(IAPI3K类调节亚基)基因敲除株被产生，导致髓系细胞IAPI3K类活性降低97%。我们推测，p21ras类IAPI3K通路的过度激活改变了NF1-/-造血细胞的增殖和存活，并有助于NF1-/-细胞移植小鼠的MPD的进展。为了验证这一假设，我们将利用Nf1+/-和p85pha基因敲除小鼠的基因杂交进行实验。其目的是：1)测试IAPI3K类基因的过度激活是否通过改变特定的信号通路而导致由NF1-/-干细胞重组的小鼠的MPD；2)检测IAPI3K类基因失活如何改变承诺的、多潜能的和原始的NF1+/+和NF1-/-祖细胞的增殖和存活；3)研究神经纤维蛋白和IAPI3K类基因如何调节体内表型特定造血细胞的细胞周期进程和存活。

DESCRIPTION (provided by applicant): Neurofibromatosis type 1 (NF1) is a genetic disorder caused by mutations in the NF1 tumor suppressor gene. Children with NF1 are predisposed to developing juvenile myelomonocytic leukemia (JMML). Neurofibromin, the protein product of NF1, is a negative regulator of p21ras activity. Though Nf1 -/- mice die in utero, mice reconstituted with Nf1 -/- fetal stem cells develop a myeloproliferative disease (MPD) similar to JMML in NF1 patients. However, alterations in p21ras signaling pathways in NF1 deficient cells responsible for MPD are unknown. Utilizing PI-3 kinase (PI3K) inhibitors, we have preliminary data implicating hyperactivation of the p21ras-PI3K pathway as responsible for the hyperproliferation and increased survival of Nf1 -/- cells. However, interpretation of results using PI3K inhibitors is limited because there are four classes of PI3K, and inhibitors inactivate all classes. Here we propose genetic experiments to determine whether hyperactivation of class IAPI3K alters the growth of Nf1 -/- hematopoietic cells. Our rationale for studying class IAPI3K in Nf1-/- cells is twofold. First, in contrast to other PI3Ks, all class IAPI3K catalytic subunits contain a p21ras-binding domain. Second, p21ras interacts with these subunits to augment kinase activity in vitro, and no evidence exists to show that p21ras augments the activity of other PI3K classes. Recently, a p85alpha (a regulatory subunit of class IAPI3K) knockout strain was generated which results in a 97% reduction in class IAPI3K activity in myeloid cells. We hypothesize that hyperactivation of the p21ras-class IAPI3K pathway, alters the proliferation and survival of Nf1 -/- hematopoietic cells and contributes to the progression of MPD in mice transplanted with Nf1 -/- cells. To test this hypothesis, we will conduct experiments utilizing a genetic intercross of Nf1 +/- and p85alpha knockout mice. The aims are: 1) To test whether hyperactivation of class IAPI3K contributes to MPD in mice reconstituted with Nf1 -/- stem cells by altering specific signaling pathways, 2) To examine how genetic inactivation of class IAPI3K alters the proliferation and survival of committed, multipotential, and primitive Nf1 +/+ and Nf1 -/- progenitor cells, 3) To examine how neurofibromin and class IAPI3K regulate cell cycle progression and survival of phenotypically defined hematopoietic cells in vivo.