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

描述（申请人提供）：1型神经纤维瘤病（NF 1）是一种由NF 1肿瘤抑制基因突变引起的遗传性疾病。 NF 1患儿易发生青少年粒单核细胞白血病（JMML）。 神经纤维蛋白，NF 1的蛋白产物，是p21 ras活性的负调节剂。 尽管Nf 1-/-小鼠在子宫内死亡，但用Nf 1-/-胎儿干细胞重建的小鼠发展出与NF 1患者中的JMML相似的骨髓增殖性疾病（MPD）。 然而，在NF 1缺陷细胞中负责MPD的p21 ras信号通路的改变是未知的。 利用PI-3激酶（PI 3 K）抑制剂，我们有初步的数据暗示p21 ras-PI 3 K通路的过度活化负责Nf 1-/-细胞的过度增殖和存活增加。 然而，使用PI 3 K抑制剂对结果的解释是有限的，因为有四类PI 3 K，而抑制剂涵盖所有类别。 在这里，我们提出了遗传实验，以确定类IAPI 3 K的过度激活是否改变Nf 1-/-造血细胞的生长。 我们在Nf 1-/-细胞中研究IAPI 3 K类的理由是双重的。 首先，与其他PI 3 K相反，所有IAPI 3 K类催化亚基都含有p21 ras结合结构域。 第二，p21 ras与这些亚基相互作用以增强体外激酶活性，并且没有证据表明p21 ras增强其他PI 3 K类的活性。 最近，产生了p85 α（IAPI 3 K类的调节亚基）敲除菌株，其导致骨髓细胞中IAPI 3 K类活性降低97%。 我们推测p21 ras类IAPI 3 K通路的过度激活改变了Nf 1-/-造血细胞的增殖和存活，并有助于移植Nf 1-/-细胞的小鼠中MPD的进展。 为了验证这一假设，我们将利用Nf 1 +/-和p85 α基因敲除小鼠的遗传杂交进行实验。 其目标是：1）为了测试IAPI 3 K类的超活化是否通过改变特定的信号传导途径而在用Nf 1-/-干细胞重建的小鼠中有助于MPD，2）为了检查IAPI 3 K类的遗传失活如何改变定型的、多能的和原始的Nf 1 +/+和Nf 1-/-祖细胞的增殖和存活，3）研究神经纤维蛋白和IAPI 3 K类如何在体内调节表型确定的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.