Identify OTX2-interacting proteins repressing differentiation in medulloblastoma

描述(申请人提供)：髓母细胞瘤是儿童最常见的脑部恶性肿瘤，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，目前的放化疗损害了儿童的发育，并造成了长期的不良影响。因此，需要根据髓母细胞瘤的分子特征进行靶向治疗，以最大限度地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/或基因扩增。它在超过60%的髓母细胞瘤中过表达。我们先前的研究已经确定OTX2是一种转录抑制因子，是髓母细胞瘤细胞中肌源性和神经细胞分化的守门人。其同源盒区(HD)显示出很强的抑制活性，使OTX2在转录水平上受到抑制。在髓母细胞瘤中，OTX2的表达缺失与肿瘤细胞群的肌源性分化有关。髓母细胞瘤是髓母细胞瘤的一种亚型，其分化的肌源性细胞与肿瘤细胞混合。在原位髓母细胞瘤异种移植模型中，shRNA诱导的OTX2基因敲除可显著延长存活时间。由于60个氨基酸的OTX2-HD具有显著的抑制活性，并且它缺乏与转录调控相关的功能基序，我们推测这种转录抑制是与表观遗传调控相互作用的结果。因此，我们建议测试潜在的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。我们建议进行体内交联/免疫纯化/串联质谱仪来鉴定OTX2-HD的结合伙伴。作为补充策略，我们建议另外使用裂解泛素酵母双杂交系统在D425髓母细胞瘤cDNA文库中寻找OTX2-HD的新的相互作用伙伴。随后，我们将分析和验证髓母细胞瘤细胞体内和体外的相互作用蛋白，并研究这些结合蛋白的功能。综上所述，了解OTX2的分子机制将促进我们开发靶向治疗的知识，从而启动OTX2抑制髓母细胞瘤的分化。这一目标可以通过确定与OTX2-HD相关的表观遗传调节因子的抑制剂和操纵与OTX2介导的抑制分化相关的潜在相互作用伙伴来实现，这是本研究的长期目的。

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.