RNAi screen of the glioblastoma stem cell kinome under hypoxia and normoxia

描述（申请人提供）：胶质母细胞瘤是一种高度侵袭性的脑肿瘤，平均生存时间仅为14个月。越来越多的证据表明，特别是脑肿瘤和胶质母细胞瘤是由癌症干细胞亚群驱动的。最近有可能在培养中分离和持续维持这些假定的肿瘤干细胞。这些细胞生长为神经球，并保留分化为神经元和星形胶质细胞的能力，尽管它们是高度致瘤性的小鼠异种移植物。我们已经开发了使用RNAi高通量筛选这些GBM干细胞的方法。在这里，我们建议全面确定在常氧和缺氧条件下胶质母细胞瘤干细胞生长和存活所需的激酶。我们将把重点放在激酶上，因为它们是可用药的靶点，有很多抑制剂已经可用，其中一些已经取得了临床成功。我们的第一个目标将是对人类kinome进行shRNA筛选，以确定胶质母细胞瘤干细胞系生长和存活通常需要的激酶。我们的初步结果表明，肿瘤干细胞生长所需的激酶与传统血清培养细胞系生长所需的激酶不同。因此，这些筛选可能会发现胶质母细胞瘤治疗的新激酶靶点。此外，胶质母细胞瘤是一种已知的具有低氧微环境的肿瘤，这可能会限制传统疗法的疗效。我们的证据表明，胶质母细胞瘤干细胞实际上在缺氧条件下茁壮成长。我们的初步数据还表明，在常氧条件下，肿瘤干细胞生长需要一些激酶，而在缺氧条件下则不需要，反之亦然。因此，确定在缺氧条件下生长和存活所需的激酶对于消除肿瘤中的癌症干细胞至关重要。因此，我们将在缺氧和常氧条件下筛选这些GBM干细胞。在第二个目标中，我们将研究在RNAi筛选中鉴定的激酶的细胞功能。此外，我们将确定筛选中发现的关键激酶是否在原发性人胶质母细胞瘤中实际上被激活，并确定在小鼠异种移植物中抑制这些激酶是否会抑制肿瘤生长。这些研究的结果应该确定并优先考虑治疗胶质母细胞瘤的新激酶靶点。

DESCRIPTION (provided by applicant): Glioblastomas are highly aggressive brain tumors with a mean survival time of only 14 months. There is increasing evidence that brain tumors and glioblastomas in particular are driven by a subpopulation of cancer stem cells. It has recently been possible to isolate and maintain continuously in culture these putative tumor stem cells. These cells grow as neurospheres and retain the ability to differentiate into neurons and astrocytes despite being highly tumorigenic mouse xenografts. We have developed methods for high throughput screening of these GBM stem cells using RNAi. Here we propose to comprehensively identify kinases that are required for the growth and survival of glioblastoma stem cells under normoxic and hypoxic conditions. We will focus on kinases since they are druggable targets for which a great many inhibitors are already available and for which several have already been clinical successes. Our first aim will be to perform shRNA screens of the human kinome to identify kinases commonly required for the growth and survival of glioblastoma stem cell lines. Our preliminary results indicate that the kinases required for tumor stem cell growth are distinct from the kinases required for growth of traditional serum grown cell lines. Thus, these screens are likely to uncover new kinase targets for glioblastoma therapy. Moreover, glioblastomas are known to be tumors that have hypoxic microenvironments that can limit the efficacy of traditional therapies. Our evidence indicates that glioblastoma stem cells actually thrive under hypoxia. Our preliminary data also indicates that there are kinases that are required for tumor stem cell growth under normoxic conditions, but not hypoxic conditions and vice versa. Identifying the kinases that are required for growth and survival under hypoxic conditions is thus critical for elimination of cancer stem cells in the tumor. Thus, we will screen these GBM stem cells under both hypoxic and normoxic conditions. In the second aim, we will investigate the cellular functions of kinases identified in the RNAi screens. In addition, we will determine whether key kinases identified in the screen are in fact activated in primary human glioblastomas, and determine whether inhibition of these kinases in mouse xenografts will inhibit tumor growth. Results of these studies should identify and prioritize new kinase targets for treatment of glioblastoma.