Identify OTX2-interacting proteins repressing differentiation in medulloblastoma

鉴定抑制髓母细胞瘤分化的 OTX2 相互作用蛋白

• 批准号: 8768857
• 负责人: Renyuan Bai
• 金额: $ 8.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8768857
• 关键词: Address; Adverse effects; Affinity; Amino Acids; Apoptosis; Binding; Binding Proteins; Biological Assay; Brain; Cancer Etiology; Cell Cycle Arrest; Cell Nucleus; Cells; Cerebellum; Cessation of life; Chemicals; Child; Child Development; Childhood; Co-Immunoprecipitations; Complex; DNA Binding; DNA Methyltransferase; DNA Methyltransferase Inhibitor; DNA Modification Methylases; DNA Sequence; Development; Enhancers; Enzymes; Epigenetic Process; Exhibits; Gatekeeping; Genes; Genetic Transcription; Goals; Histologic; Histone Deacetylase; Histones; Homeobox; In Vitro; Knowledge; Late Effects; Lead; Libraries; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Medullomyoblastoma; Membrane; Methods; Methylation; Molecular; Morbidity - disease rate; Muscle; Muscle Cells; Nature; Neurocognitive; Neuronal Differentiation; Neurons; Oncogenic; Patients; Population; Primitive Neuroectodermal Tumor; Procedures; Protein Binding; Proteins; Reporter; Reporter Genes; Reporting; Repression; Series; Small Interfering RNA; System; Testing; Therapeutic; Transcription Coactivator; Transcription Repressor/Corepressor; Transcriptional Regulation; Ubiquitin; Undifferentiated; Validation; Xenograft Model; Yeasts; base; cDNA Library; chemoradiation; crosslink; gene repression; high risk; histone methyltransferase; immunoaffinity chromatography; in vivo; inhibitor/antagonist; medulloblastoma; neoplastic cell; neurodevelopment; novel; overexpression; prevent; protein crosslink; public health relevance; screening; small hairpin RNA; tandem mass spectrometry; therapeutic development; transcription factor; tumor; yeast two hybrid system

DESCRIPTION (provided by applicant): Medulloblastoma, the most common type of pediatric brain malignancy, is an aggressive primitive neuroectodermal tumor arising from the cerebellum. It presents a significant cause of cancer-related death in children and current treatment of radio-chemotherapy impairs children's development and causes long-term adverse effects. Therefore, targeted therapies based on the molecular features of medulloblastoma are needed to minimize the treatment-related side effects in children. The transcription factor OTX2 is overexpressed and/or genomically amplified in most medulloblastomas. It is overexpressed in over 60% of medulloblastoma. Our previous study has identified OTX2 as a transcriptional repressor and a gatekeeper of myogenic and neuronal differentiation in medulloblastoma cells. The strong repressor activity exhibited by its homeobox domain (HD) renders OTX2 transcriptionally repressive. In medullomyoblastoma, a subtype of medulloblastoma with differentiated myogenic cells mixed with tumor cells, we have linked the loss of expression of OTX2 to the myogenic differentiation in tumor cell populations. Inducible knockdown of OTX2 by shRNA led to significant extension of survival in an orthotopic medulloblastoma xenograft model. Owing to the remarkable repressor activity displayed by the 60 amino acid OTX2-HD and its lack of functional motif related to transcriptional regulators, we hypothesize that this transcriptional repression is the result of interactions with epigenetic regulators. We therefore propose to test inhibitors of potential epigenetic regulators to characterize the nature of OTX2-HD mediated repression. We propose to perform in vivo crosslinking/immuno-purification/tandem mass spectrometry to identify binding partners of OTX2-HD. As a complementary strategy, we suggest additionally to use split-ubiquitin yeast two-hybrid system to identify novel interacting partners of OTX2-HD in a D425 medulloblastoma cDNA library. Subsequently, we will analyze and validate the interacting proteins in vitro and in vivo in medulloblastoma cells, and study the functionality of those binding proteins. In summary, understanding the molecular mechanism of OTX2 will advance our knowledge to develop targeted therapies that initiate the differentiation suppressed by OTX2 in medulloblastoma. This goal can be achieved by identifying the inhibitors of epigenetic regulators associated with OTX2- HD and by manipulating the potential interacting partners relevant in OTX2-mediated suppression of differentiation, which is the long term purpose of the study.

描述（申请人提供）：髓母细胞瘤是最常见的儿童脑恶性肿瘤，是一种起源于小脑的侵袭性原始神经外胚层肿瘤。它是儿童癌症相关死亡的一个重要原因，目前的放化疗治疗损害儿童发育并造成长期不良影响。因此，需要根据成神经管细胞瘤的分子特征进行靶向治疗，以尽量减少儿童治疗相关的副作用。转录因子OTX2在大多数髓母细胞瘤中过度表达和/或基因组扩增。它在60%以上的髓母细胞瘤中过表达。我们之前的研究已经发现OTX2在成神经管细胞瘤细胞中是一种转录抑制因子和肌源性和神经元分化的守门人。其同源盒结构域（HD）表现出强烈的抑制活性，使OTX2具有转录抑制作用。髓母细胞瘤是髓母细胞瘤的一种亚型，分化的肌源性细胞与肿瘤细胞混合，我们已经将OTX2的表达缺失与肿瘤细胞群的肌源性分化联系起来。在原位髓母细胞瘤异种移植模型中，通过shRNA诱导敲低OTX2可显著延长存活时间。由于60个氨基酸的OTX2-HD表现出显著的抑制活性，并且缺乏与转录调节因子相关的功能基序，我们假设这种转录抑制是与表观遗传调节因子相互作用的结果。因此，我们建议测试潜在表观遗传调节因子的抑制剂，以表征OTX2-HD介导的抑制的性质。我们建议采用体内交联/免疫纯化/串联质谱法来鉴定OTX2-HD的结合伙伴。作为补充策略，我们建议另外使用分裂-泛素酵母双杂交系统来鉴定D425髓母细胞瘤cDNA文库中OTX2-HD的新相互作用伙伴。随后，我们将在体外和体内对成神经管细胞瘤细胞的相互作用蛋白进行分析和验证，并研究这些结合蛋白的功能。总之，了解OTX2的分子机制将提高我们的知识，开发靶向治疗，启动髓母细胞瘤中OTX2抑制的分化。这一目标可以通过鉴定与OTX2- HD相关的表观遗传调节因子的抑制剂，以及通过操纵OTX2介导的分化抑制相关的潜在相互作用伙伴来实现，这是本研究的长期目的。