The Role of Bcl2L12 in the Genesis of Malignant Glioma

描述(申请人提供)：多形性胶质母细胞瘤(GBM)是恶性胶质瘤最具侵袭性的表现，其特征是微血管增殖、坏死，对现有治疗方法极端抵抗，以及神经学破坏性过程，通常在确诊后12个月内死亡。主要的概念差距仍然存在于机制层面，包括经典的遗传损伤如何对这些表型方面做出贡献。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进行了癌基因组分析，以努力获得对GBM发育的遗传事件的更全面的看法。在鉴定染色体19q上的增益区的过程中，我们发现Bcl2L12(对于Bcl2-like-12)是一种胶质瘤癌基因，在几乎所有的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，在原代培养的星形细胞中作为一种抗凋亡蛋白发挥作用。由于已知在线粒体后水平抑制细胞凋亡可以阻止细胞凋亡但促进坏死，Bcl2L12在GBM中的过度表达可能为这种疾病生物学中的一个主要悖论--细胞凋亡抵抗和大量坏死--提供一个合理的解释，并指出Bcl2L12上调是恶性胶质瘤的关键进展事件。除了胞内caspase-3/7抑制活性外，Bcl2L12还存在于核质中，在那里它与P53相互作用并阻断P53介导的反式激活。为了在基于细胞培养的分析中更详细地分析Bcl2L12调控的通路，并在体内对这些发现进行遗传学验证，这项拨款提案旨在通过详细的结构-功能分析进一步阐明Bcl2L12‘S致癌活性的分子基础。我们将使用基于细胞培养的分析和原位SCID移植瘤模型，使用基因工程神经干细胞和成熟的皮质星形胶质细胞来分析细胞凋亡和P53的调节活性。此外，我们将通过对P53介导的转录调控的详细研究，深入研究定义Bcl2L12另一致癌活性的P53抑制的分子机制。最后，将使用有条件的Bcl2L12基因敲除小鼠来评估Bcl2L12失活对GBM发育的影响，这可能是一个体内平台，在此平台上评估Bcl2L12在恶性胶质瘤治疗中的药理失活后果。 在治疗高级别胶质瘤方面仍然缺乏成功，促使人们重新评估胶质瘤药物开发的方方面面。这项赠款提案旨在通过完善对疾病的分子理解以及开发更准确的体外和体内胶质瘤模型系统，专门解决目前在开发有效的胶质瘤治疗方法方面尚未得到满足的需求。

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.