Targeting the Novel PI5P4K Pathway to Induce Glioblastoma Senescence

描述(申请人提供)：细胞衰老是一种肿瘤抑制机制。然而，多形性胶质母细胞瘤(GBM)是最恶性的原发脑肿瘤，中位生存期约一年，对衰老具有高度的抵抗力。迫切需要新的治疗方式和药物，使GBM对衰老敏感，以改善患者的预后。一个PI5P4K家族将磷脂酰肌醇5-磷酸(PI5P)磷酸化，并将其转化为磷脂酰肌醇4，5-磷酸(PI[4，5]P2)，以调节磷脂酰肌醇信号转导。在初步研究中，我的实验室发现PI5P4Kβ直接与鸟嘌呤核苷酸结合。我们的X射线结构分析表明，PI5P4Kβ利用GTP作为首选的磷酸供体，而不是ATP。动力学分析还预测，生理水平的GTP控制PI5P4Kβ的活性。重要的是，我们发现细胞内GTP水平在抑制基底膜衰老中起着关键作用，而PI5P4Kβ基因敲除会触发基底膜细胞的衰老。我们已经确定了一种在体外和体内都能抑制PI5P4K活性的小化合物。PI5P4K化学抑制剂处理可诱导GBM细胞衰老。这些结果表明，PI5P4Kβ抑制剂可能在预防或治疗基底膜方面有一定的作用。本研究的目的是确定一种新型的依赖于GTP的蛋白激酶PI5P4Kβ在调节基底膜衰老中的意义，并确定抑制PI5P4β是否能够诱导基底膜衰老并降低其在体内外的致瘤活性。为了验证这一假说，我们将利用体外细胞培养和临床相关的胶质母细胞瘤原位种植模型(Aim 1)，通过shRNA介导的PI5P4Kβ抑制和药物抑制来确定PI5P4Kβ在基底膜细胞生长中的作用。我们将使用生化和生物学分析来确定PI5P4Kβ(目标2)检测GTP的机制。我们将通过表征PI5P4Kβ下游效应器来确定PI5P4Kβ是如何调控衰老的(目标3)。总之，这些研究将揭示靶向PI5P4Kβ诱导胶质母细胞瘤细胞衰老和生长停滞的新机制和机会。

DESCRIPTION (provided by applicant): Cellular senescence is a tumor suppressor mechanism. However glioblastoma multiforme (GBM), the most malignant primary brain tumor with a median survival of about one year, is highly resistant to senescence. There is a critical need for new therapeutic modalities and agents that sensitize GBM to senescence to improve patient outcome. A family of PI5P4K phosphorylates phosphatidylinositol 5-phosphate (PI5P) and converts it to phosphatidylinositol 4,5-phosphate (PI[4,5]P2) to regulate phosphoinositide signaling. In the preliminary studies, my laboratory found that PI5P4Kβ binds directly to guanine nucleotides. Our X-ray structural analyses show that PI5P4Kβ utilizes GTP as the preferred phospho-donor, as opposed to ATP. Kinetic analyses also predict that physiological GTP levels control the PI5P4Kβ activity. Importantly, we have found that cellular GTP levels play critical rol in suppressing GBM senescence and that PI5P4Kβ knockdown triggers senescence of GBM cells. We have identified a small chemical compound that inhibits PI5P4K activity in vitro and in vivo. The treatment of the PI5P4K chemical inhibitor induces senescence of GBM cells. These results indicate that inhibitors of PI5P4Kβ could be useful in preventing or treating GBM. The goal of this proposal is to determine the significance of a novel type of GTP-dependent kinase, PI5P4Kβ, in regulating GBM senescence, and to determine whether inhibition of PI5P4β could induce GBM senescence and reduce its tumorigenic activity in vitro and in vivo. To test the hypothesis, we will determine the role of PI5P4Kβ in GBM cell growth by shRNA-mediated PI5P4Kβ knockdown and pharmacological inhibition using in vitro cell culture and a clinically relevant orthotopic implantation model of glioblastoma (Aim 1). We will use biochemical and biological analyses to determine the mechanism of GTP detection by PI5P4Kβ (Aim 2). We will determine how PI5P4Kβ regulates senescence by characterizing PI5P4Kβ downstream effectors (Aim 3). Collectively, these studies will lead to reveal the novel mechanism and opportunity for inducing senescence and growth arrest of glioblastoma cells by targeting PI5P4Kβ.