The Role of Bcl2L12 in the Genesis of Malignant Glioma

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）是恶性胶质瘤中最具侵袭性的表现，其特征是微血管增生、坏死，对所有现有治疗方式都具有极强的耐药性，并且通常在诊断后12个月内出现神经系统破坏性病程，最终导致死亡。主要的概念差距仍然存在于机制水平上，包括经典遗传病变如何促成这些表型方面。为了解决这个问题，我们通过阵列- cgh和表达谱对大量人类GBM进行了肿瘤基因组分析，以获得更全面的GBM发展背后的遗传事件。在描述染色体19q上的一个增加区域的过程中，我们发现Bcl2L12（对于bcl2 - like12）是一种胶质瘤癌基因，在几乎所有GBM样本中都过表达，但在低级别疾病和正常组织中低表达或不表达。广泛的生化研究已经证实Bcl2L12通过抑制线粒体后caspase-3和caspase-7的激活，在原代星形细胞培养中发挥抗凋亡蛋白的作用。由于已知线粒体后水平的细胞凋亡抑制可阻断细胞凋亡但促进坏死，Bcl2L12在GBM中的过表达可能为该疾病生物学中的一个主要悖论提供了合理的解释-细胞凋亡抵抗但呈花状坏死-并指出Bcl2L12上调是恶性胶质瘤的关键进展事件。除了胞质内的caspase-3/7抑制活性外，Bcl2L12还存在于核质中，与p53相互作用并阻断p53介导的转激活。为了在基于细胞培养的实验中更详细地分析Bcl2L12调控的途径，并在体内对这些发现进行遗传验证，本资助申请旨在通过详细的结构-功能分析进一步阐明Bcl2L12致癌活性的分子基础。我们将采用基于细胞培养的实验和使用基因工程神经干细胞和成熟皮层星形胶质细胞的原位SCID外植体肿瘤模型来解剖细胞凋亡和p53调节活性。此外，我们将通过对p53介导的转录调控的详细研究，深入表征p53抑制的分子机制，该机制定义了Bcl2L12的另一种致癌活性。最后，Bcl2L12失活对GBM发展的影响将通过条件Bcl2L12敲除小鼠进行评估，该小鼠可能作为体内平台，评估Bcl2L12药物失活对恶性胶质瘤治疗的影响。

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM), the most aggressive manifestation of malignant gliomas, are characterized by microvascular proliferation, necrosis, extreme resistance to all extant therapeutic modalities and a neurologically destructive course culminating in death often within 12 months of diagnosis. Major conceptual gaps remain on the mechanistic level including how the classical genetic lesions contribute to these phenotypic aspects. To address this issue, we performed oncogenomic analyses of a large panel of human GBMs by array-CGH and expression profiling in an effort to gain a more comprehensive view of the genetic events underlying GBM development. In the course of characterizing a region of gain on chromosome 19q, we identified Bcl2L12 (for Bcl2-Like-12) as a glioma oncogene that is over-expressed in virtually all GBM samples, yet low or absent in low-grade disease and normal tissue. Extensive biochemical studies have established that Bcl2L12 functions as an anti-apoptotic protein in primary astrocytic cultures by inhibiting post-mitochondrial caspase-3 and caspase-7 activation. Since inhibition of apoptosis at the post- mitochondrial level is known to block apoptosis but promotes necrosis, Bcl2L12 over-expression in GBM may provide a rational explanation for a prime paradox in the biology of this disease - apoptosis resistance yet florid necrosis - and points to Bcl2L12 up-regulation as a key progression event in malignant glioma. In addition to its cytosolic caspase-3/7 inhibitory activity, Bcl2L12 resides in the nucleoplasm where it interacts with p53 and blocks p53-mediated transactivation. To analyze Bcl2L12-modulated pathways in cell culture- based assays in more detail and to genetically validate these findings in vivo, this grant proposal aims to elucidate further the molecular basis of Bcl2L12's oncogenic activity through detailed structure-function analyses. We will employ cell culture-based assays and orthotopic SCID explant tumor models using genetically engineered neuronal stem cells and mature cortical astrocytes to dissect apoptosis- and p53 modulatory activities. Furthermore, we will characterize in depth the molecular mechanism of p53 inhibition that defines yet another oncogenic activity of Bcl2L12 through detailed studies of p53-mediated transcriptional regulation. Finally, the consequences of Bcl2L12 inactivation for the development of GBM will be assessed using conditional Bcl2L12 knockout mice that may serve as an in vivo platform on which to assess consequence of pharmacological inactivation of Bcl2L12 for malignant glioma therapy. The continued lack of success in treating high-grade gliomas has prompted a reevaluation of all aspects of glioma drug development. This grant proposal aims to specifically address the currently unmet needs in the development of effective glioma therapies by refining the molecular understanding of the disease and by developing more accurate in vitro and in vivo glioma model system.