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

描述（由申请人提供）：1型神经纤维瘤病（NF1）是一种由NF1肿瘤抑制基因突变引起的遗传性疾病。患有NF1的儿童易患幼年粒细胞白血病（JMML）。神经纤维蛋白是NF1的蛋白产物，是p21ras活性的负调节因子。虽然Nf1 -/-小鼠在子宫内死亡，但用Nf1 -/-胚胎干细胞重建的小鼠在Nf1患者中出现类似JMML的骨髓增生性疾病（MPD）。然而，导致MPD的NF1缺陷细胞中p21ras信号通路的改变尚不清楚。利用PI-3激酶（PI3K）抑制剂，我们有初步数据表明p21ras-PI3K通路的过度激活是Nf1 -/-细胞过度增殖和存活率增加的原因。然而，使用PI3K抑制剂对结果的解释是有限的，因为有四类PI3K，抑制剂灭活所有类。在这里，我们提出了遗传学实验来确定IAPI3K类的过度激活是否会改变Nf1 -/-造血细胞的生长。我们研究Nf1-/-细胞中的IAPI3K类有两个理由。首先，与其他pi3k相比，所有IAPI3K类催化亚基都含有p21ras结合结构域。其次，p21ras在体外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p85alpha （IAPI3K类的调控亚基）敲除菌株产生，导致髓细胞中IAPI3K类活性降低97%。我们假设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MPD的进展。为了验证这一假设，我们将利用Nf1 +/-和p85alpha基因敲除小鼠的遗传交叉进行实验。目的是：1)通过改变特定的信号通路，测试IAPI3K类的过度激活是否有助于Nf1 -/-干细胞重组小鼠的MPD； 2)研究IAPI3K类的基因失活如何改变固定的、多电位的和原始的Nf1 +/+和Nf1 -/-祖细胞的增殖和存活；3)研究神经纤维蛋白和IAPI3K类如何调节体内表型定义的造血细胞的细胞周期进展和存活。

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.