Targeting the Kinome in Neurofibromatosis type 1

描述(申请人提供)：神经纤维瘤病1型(NF1)是一种常见的遗传性疾病综合征，由NF1基因的胚系突变引起。大约三分之一的NF1患者发展为弥漫性丛状神经纤维瘤，可能会转化为恶性周围神经鞘瘤--一种经常致命的癌症。值得注意的是，在人类肿瘤和NF1小鼠模型中，神经纤维瘤几乎总是包含NF1缺失的雪旺细胞和NF1杂合的肥大细胞。将这种倾向于NF1的小鼠移植到野生型骨髓中可以防止肿瘤的发生，这意味着杂合的骨髓衍生细胞(如肥大细胞)是发病过程中必需的组成部分，而靶向雪旺细胞或肥大细胞中的信号通路可能对治疗有益。NF1基因编码一种被称为神经纤维素的大蛋白，具有针对RAS的GTP酶激活蛋白(GAP)活性。NF1基因的完全或半合子缺失导致雪旺细胞和肥大细胞中K-ras活性增加，伴随而来的是下游效应因子的激活，从而促进细胞增殖和细胞形状和运动的变化。最近，我们发现p21激活的蛋白激酶(PAK)在K-ras信号转导中发挥重要作用，特别是在TE、Erk和Akt信号通路的激活中。随着临床级别的小分子Pak抑制剂最近出现，我们提出的实验与NF1综合征的治疗应用之间存在直接联系，或许也适用于由获得性、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缺陷细胞中数百个蛋白激酶的活性，证实了已知的K-ras激活的信号活性(例如，升高的 Pak、Erk和Akt/mTOR通路的活性)，但也发现了靶向治疗的新的潜在候选者。在这项建议中，我们假设K-ras下游ERK和Akt/mTOR通路激活所需的PAK是唯一适合作为NF1驱动的丛状神经纤维瘤和恶性周围神经鞘瘤治疗的靶点。此外，我们假设，如果出现对抗PAK药物的耐药性，可以使用完整的基因组分析来识别可能的“逃逸”路线，从而实现更有效的联合治疗。因此，我们 提出三个目标：1)我们将确定NF1-/-雪旺细胞和肥大细胞中动态组的基线状态，以及在MEK、Akt/mTOR或Pak抑制的压力下，该动态组如何重新编程；2)我们将使用NF1和Pak的小鼠模型来确定Pak在NF1相关肿瘤中发挥作用的细胞学基础；以及3)我们将确定特定的小分子Pak抑制剂在NF1小鼠模型和异种移植瘤中的疗效，并评估此类药物在体内引发的信号反应。

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.