Targeting the Kinome in Neurofibromatosis type 1

描述（由申请人提供）：1型神经纤维瘤病（NF1）是一种常见的遗传性疾病综合征，由NF1基因的种系突变引起。大约三分之一的NF1患者发展为弥漫性丛状神经纤维瘤，可转变为恶性周围神经鞘肿瘤，这是一种经常致命的癌症。值得注意的是，在人类肿瘤和小鼠NF1模型中，神经纤维瘤几乎无一例外地含有NF1缺失的雪旺细胞和NF1杂合肥大细胞。将这种nf1易感小鼠与野生型骨髓移植可防止肿瘤发生，这意味着杂合的骨髓来源细胞（如肥大细胞）是发病机制中必需的组成部分，并且靶向雪旺细胞或肥大细胞中的信号通路可能具有治疗益处。NF1基因编码一种称为神经纤维蛋白的大蛋白，具有GTPase激活蛋白（GAP）对Ras的活性。NF1基因的完全或半合子缺失会导致雪旺细胞和肥大细胞中K-ras活性的增加，并伴随下游效应物的激活，从而促进增殖，改变细胞形状和运动。最近，我们发现p21活化激酶（Paks）在K-ras信号传导中起着至关重要的作用，特别是在Erk和Akt通路的激活中。随着临床级小分子Pak抑制剂的出现，我们提出的实验与NF1综合征的治疗应用有直接的联系，也可能是由获得性体细胞NF1突变驱动的其他癌症。此外，我们使用了一种新的磷酸化蛋白质组学方法来分析nf1缺陷细胞中数百种蛋白激酶的活性，证实了已知的k -ras激活的信号活性(例如，升高

DESCRIPTION (provided by applicant): Neurofibromatosis type 1 (NF1) is a common inherited disease syndrome caused by germline mutations in the NF1 gene. About one third of NF1 patients develop diffuse, plexiform neurofibromas that can transform to malignant peripheral nerve sheath tumors - a cancer that is frequently fatal. Remarkably, in human tumors and in mouse models of NF1, neurofibromas almost invariably contain NF1-null Schwann cells and NF1-heterozygous mast cells. Transplanting such NF1-prone mice with wild-type bone marrow prevents tumorigenesis, implying that heterozygous bone marrow-derived cells such as mast cells are a required component in pathogenesis, and that targeting signaling pathways in either Schwann cells or in mast cells might be of therapeutic benefit. The NF1 gene encodes a large protein, termed Neurofibromin, with GTPase Activating Protein (GAP) activity towards Ras. Complete or hemizygous loss of the NF1 gene leads to increased K-ras activity in both Schwann cells and mast cells, with concomitant activation of downstream effectors that promote proliferation and changes in cell shape and movement. Recently, we have shown that p21-activated kinases (Paks) play a vital role in K-ras signaling, in particular in the activation of te Erk and Akt pathways. As clinical-grade small molecules Pak inhibitors have recently emerged, there is a direct line from our proposed experiments to therapeutic application in NF1 syndrome, and perhaps also in other cancers driven by acquired, somatic NF1 mutations. In addition, we have used a new phospho-proteomic method to analyze the activity of hundreds of protein kinases in NF1-deficient cells, confirming known K-ras-activated signaling activity (e.g., elevated activity of Pak, Erk, and Akt/mTOR pathways) but also uncovering new potential candidates for targeted therapy. In this proposal, we postulate that Paks, which are required both for Erk and Akt/mTOR pathway activation downstream of K-ras, are uniquely suited as targets for therapy in NF1-driven plexiform neurofibromas and malignant peripheral nerve sheath tumors. Further, we posit that, should resistance emerge to anti-Pak agents, whole kinome analysis can be used to identify likely "escape" routes, allowing for more effective combinatorial therapy. Accordingly, we propose three aims: 1) We will determine the baseline status of the kinome in NF1-/- Schwann cells and mast cells, and how the kinome reprograms under pressure of Mek, Akt/mTOR, or Pak inhibition; 2) We will use mouse models of NF1 and Pak to determine the cellular basis for Pak's function in NF1-related tumors; and 3) We will determine the efficacy of specific small molecule Pak inhibitors in NF1 mouse models and xenografts, and assess the signaling responses elicited by such agents in vivo.