F-box ubiquitin ligases destabilize neurofibromin

项目总结/摘要 NF 1肿瘤抑制基因的突变导致1型神经纤维瘤病（NF 1），这是人类最常见的 遗传性癌症易感综合征患有NF 1的个体患有广泛的恶性肿瘤， 非恶性临床表现包括丛状神经纤维瘤（PN），复杂的癌前病变 其影响25-40%的NF 1患者并导致主要的终身发病率和死亡率。NF 1基因编码 neurofibromin，一种p21 ras（Ras）的GTP酶激活蛋白（GAP）。我们先前确定， Nf 1单个等位基因（Nf 1 +/-）导致Nf 1患者（Nf 1 +/-）和鼠（Nf 1 +/-）骨髓细胞中Ras过度活化， 这个概念被称为单倍不足。我们建立了一个基因工程小鼠模型， 神经纤维瘤病，并证明丛状神经纤维瘤的形成需要炎性 Nf 1 +/-骨髓的贡献。此外，我们发现，下游激酶通路的遗传抑制 造血系统中的神经纤维蛋白酶可防止肿瘤发生。这项工作导致了有史以来第一次 在临床前模型和我们的II期临床试验中，这些肿瘤的成功药物治疗。 尽管取得了这一成功，但在神经纤维蛋白酶缺乏的组织中纠正Ras过度活化的补充策略 由于Ras介导的信号通路的复杂性和患者反应的异质性， 对于这些复杂肿瘤的激酶抑制剂，这些肿瘤对传统化疗完全抵抗， 放射治疗 神经纤维蛋白被磷酸化，泛素化，并在蛋白酶体降解，以响应生长因子 刺激，但很少有人知道这个过程的机制方面。具体来说，泛素连接酶 控制肿瘤驱动造血细胞中神经纤维蛋白降解的特异性因子（E3）不 知道的F-box泛素连接酶以磷酸化依赖的方式降解选定的蛋白质，导致蛋白质的磷酸化。 我们假设NF 1 E3属于F-box家族。在最初未发表的研究中， 为了准备这个应用，我们进行了RNAi筛选，鉴定了强的新候选F-box NF 1的泛素连接酶。在这里，我们建议机械地检查这些新发现的F盒蛋白质 调节离体和体内神经纤维蛋白降解。我们还建议采用一个无偏泛函 基因组学策略，以鉴定促进神经纤维蛋白的F-box依赖性降解的激酶， 泛素-蛋白酶体途径我们将采用多学科的方法来确定是否中断 我们的候选神经纤维蛋白E3在体内可以挽救神经纤维蛋白单倍不足， 神经纤维瘤的发生和发展。这些研究还将提供基本的见解， 常见但研究不足的肿瘤抑制基因。

PROJECT SUMMARY / ABSTRACT Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), the most common human genetic cancer predisposition syndrome. Individuals with NF1 suffer from a wide range of malignant and nonmalignant clinical manifestations including plexiform neurofibromas (PN), complex precancerous lesions which affect 25-40% of NF1 patients and cause major lifelong morbidity and mortality. The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for p21ras (Ras). We previously determined that loss of a single allele of Nf1 (Nf1+/-) results in Ras hyperactivation in NF1 patient (NF1+/-) and murine (Nf1+/-) myeloid cells, a concept known as haploinsufficiency. We generated a genetically engineered murine model of neurofibromatosis and demonstrated that plexiform neurofibroma formation requires the inflammatory contribution of Nf1+/- bone marrow. Further, we showed that genetic inhibition of kinase pathways downstream of neurofibromin in the hematopoietic system prevents tumorigenesis. This work has led to the first ever successful pharmacological treatment of these tumors in both preclinical models and in our phase II clinical trial. Despite this success, complementary strategies to correct Ras hyperactivation in neurofibromin-deficient tissues are needed due to the complexity of Ras-mediated signaling pathways and the heterogeneity of patient response to kinase inhibitors for these complex tumors that are completely resistant to traditional chemotherapy and radiation treatment. Neurofibromin is phosphorylated, ubiquitinated, and degraded at the proteasome in response to growth factor stimulation, but little is known about the mechanistic aspects of this process. Specifically, the ubiquitin ligase specificity factor(s) (E3) that govern neurofibromin degradation in the tumor-driving hematopoietic cells are not known. The F-box ubiquitin ligases degrade selected proteins in a phosphorylation-dependent manner, leading us to hypothesize that the NF1 E3 belongs to the F-box family. In initial unpublished studies pursued in preparation for this application, we conducted an RNAi screen, which identified strong novel candidate F-box ubiquitin ligases for NF1. Here, we propose to mechanistically examine these newly identified F-box proteins modulating neurofibromin degradation ex vivo and in vivo. We also propose to employ an unbiased functional genomics strategy to identify kinase(s) that promote F-box-dependent degradation of neurofibromin via the ubiquitin-proteasome pathway. We will employ a multidisciplinary approach to determine whether disruption of our candidate neurofibromin E3s in vivo can rescue neurofibromin haploinsufficiency and prevent plexiform neurofibroma initiation and progression. These studies will also provide basic insights into the regulation of a common but understudied tumor suppressor gene.