F-box ubiquitin ligases destabilize neurofibromin

F-box 泛素连接酶破坏神经纤维蛋白的稳定性

基本信息

批准号：
10249088
负责人：
David W Clapp
金额：
$ 34.45万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10249088
关键词：
Affect Alleles Automobile Driving B-Lymphocytes Biochemical Bone Marrow Bone Marrow Transplantation Cells Chemotaxis Chemotherapy and/or radiation Clinical Complex F Box Domain F-Box Proteins Family GTPase-Activating Proteins Genes Genetic Genetically Engineered Mouse Genome Genomic approach Glioblastoma Growth Factor Hematopoietic Hematopoietic System Hematopoietic stem cells Heterogeneity Human Human Genetics Individual Inflammatory Knockout Mice Lesion Malignant - descriptor Malignant Neoplasms Malignant neoplasm of lung Mediating Metastatic Melanoma Morbidity - disease rate Mus Mutation Myeloid Cells NF1 gene Neurofibromatoses Neurofibromatosis 1 Non-Malignant Pathologic Pathway interactions Patients Pharmacological Treatment Phase II Clinical Trials Phenotype Phosphorylation Phosphotransferases Plexiform Neurofibroma Pre-Clinical Model Preparation Process Proteins RNA interference screen Radiation therapy Regulation Resistance Signal Pathway Signal Transduction Small Interfering RNA Specificity Stem Cell Factor Syndrome Testing Tissues Tumor Suppressor Genes Tumor Suppressor Proteins Ubiquitin Ubiquitination Work cancer predisposition clinically significant drug discovery functional genomics in vivo innovation insight interdisciplinary approach kinase inhibitor mast cell mortality multicatalytic endopeptidase complex novel patient response premalignant prevent recruit response screening success therapeutic candidate therapeutic target tumor tumorigenesis ubiquitin ligase ubiquitin-protein ligase

项目摘要

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.

项目概要/摘要 NF1 抑癌基因突变会导致 1 型神经纤维瘤病 (NF1)，这是人类最常见的疾病遗传性癌症易感综合症。患有 NF1 的个体患有多种恶性和非恶性临床表现，包括丛状神经纤维瘤（PN）、复杂的癌前病变它影响 25-40% 的 NF1 患者，并导致重大的终生发病率和死亡率。 NF1基因编码神经纤维蛋白，一种 p21ras (Ras) 的 GTP 酶激活蛋白 (GAP)。我们之前确定损失 Nf1 (Nf1+/-) 的单一等位基因导致 NF1 患者 (NF1+/-) 和小鼠 (Nf1+/-) 骨髓细胞 Ras 过度激活，一个称为单倍体不足的概念。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症 Nf1+/-骨髓的贡献。此外，我们发现下游激酶途径的遗传抑制造血系统中的神经纤维蛋白可预防肿瘤发生。这项工作导致了有史以来第一个在临床前模型和 II 期临床试验中成功对这些肿瘤进行药物治疗。尽管取得了这一成功，但纠正神经纤维蛋白缺陷组织中 Ras 过度激活的补充策略由于 Ras 介导的信号通路的复杂性和患者反应的异质性，需要针对这些对传统化疗完全耐药的复杂肿瘤的激酶抑制剂放射治疗。神经纤维蛋白在生长因子的作用下被磷酸化、泛素化和蛋白酶体降解刺激，但人们对这一过程的机制知之甚少。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) 不是已知。 F-box 泛素连接酶以磷酸化依赖性方式降解选定的蛋白质，从而导致我们假设 NF1 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，确定了强大的新颖候选 F-box NF1 泛素连接酶。在这里，我们建议从机制上检查这些新发现的 F-box 蛋白调节离体和体内神经纤维蛋白降解。我们还建议采用无偏函数基因组学策略来识别通过 F-box 依赖性促进神经纤维蛋白降解的激酶泛素-蛋白酶体途径。我们将采用多学科方法来确定是否会破坏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体不足并预防丛状神经纤维瘤的发生和进展。这些研究还将提供有关监管的基本见解。常见但尚未得到充分研究的肿瘤抑制基因。