Elucidation and Exploitation of GSK3 as a Novel Glioma Therapeutic Target

我们已经证明了多种GSK 3活性的小分子抑制剂， GSK 3 α/β基因下调对胶质瘤细胞生长抑制作用 生存在使用的小分子中，LY 317615由Eli Lilly Pharmaceuticals开发 作为PKC-β（PKC-b）的ATP竞争性抑制剂，抑制VEGF刺激的内皮细胞 增殖，并应用于临床前肿瘤模型，其中它表现出显着的 抗血管生成活性。鉴于其他PKC同工酶已被证明有助于 肿瘤细胞的存活和增殖，我们试图研究是否Enzavalin可以 通过抑制PKC-b活性直接对胶质瘤细胞发挥抗增殖活性。我们 发现LY 317615对神经胶质瘤细胞系具有有效的抗增殖活性， 可达到的浓度（IC 50为10 mM）。我们试图确定 LY 317615对胶质瘤细胞增殖的抑制机制。细胞周期分析执行人 LY 317615处理的U251的BrdU/PI染色显示药物诱导的G2/M期阻滞和凋亡， 早在治疗后24小时。阐明抗胶质瘤作用的机制 我们进行了基因表达谱分析，希望能识别出潜在的 PKC-b抑制的下游效应物。引人注目的是， 脑胶质瘤后近1400个mRNA转录物中的Wnt通路发生显著改变 细胞暴露于LY 317615。上调最强的基因（超过40倍）是axin 2 mRNA，Wnt负反馈环的已知组分。除了axin 2，我们还发现 至少20个基因表达的高度显著变化，如CyclinD 1， 已知是Wnt通路的下游效应物β-连环蛋白的靶点。进一步 通过药理学和遗传学手段对该途径的研究表明， 神经胶质瘤细胞系中Wnt途径的激活导致细胞死亡。具体来说，我们有 表明细胞毒性作用的效力与细胞毒性作用的降低直接相关。 GSK 3 α/β Y276/Y216的酶活性激活磷酸化，和 具有增加的酶活性-GSK 3 α S21的抑制性磷酸化。 抑制GSK 3活性导致c-MYC活性的细胞毒性依赖性增加 从而诱导Bim、bax和DR 4/DR 5的表达。GSK 3活性的下调也 导致FLIP蛋白下降和TRAIL上调。除了上调 TRAIL相关的外源性凋亡途径的组分，GSK 3的下调 活性导致细胞内葡萄糖代谢的改变，导致解离 己糖激酶（HK）II从线粒体外膜与随后的线粒体 不稳定最后，GSK 3活性的抑制导致细胞凋亡的显著降低。 细胞内核因子-κ B（NF-B）活性。因此，抑制GSK 3 活性通过多种机制导致c-MYC依赖性胶质瘤细胞死亡， 汇聚在凋亡途径上。这些数据支持GSK 3可能是 胶质瘤重要治疗靶点。基于有希望的临床前数据，我们 在复发性高级别胶质瘤患者中启动了LY 317615的临床试验

We have demonstrated that multiple small molecular inhibitors of GSK3 activity and genetic downregulation of GSK3alpha/beta significantly inhibit glioma cell survival. Among the small molecules used, LY317615 was developed by Eli Lilly Pharmaceuticals as an ATP-competitive inhibitor of PKC-beta (PKC-b) to inhibit VEGF-stimulated endothelial proliferation and applied in preclinical tumor models where it demonstrated significant anti-angiogenic activity. Given that other PKC isoenzymes have been shown to contribute to tumor cell survival and proliferation, we sought to investigate whether Enzastaurin could exert anti-proliferation activity on glioma cells directly by inhibiting PKC-b activity. We found that LY317615 exerts potent anti-proliferation activity on glioma cell lines at pharmacologically achievable concentrations (IC50 of 10mM). We sought to determine the anti-proliferative mechanism of LY317615 on glioma cells. Cell Cycle analysis preformed by BrdU/PI staining of LY317615-treated U251 revealed a drug-induced G2/M arrest and apoptosis as early as 24hr after treatment. To elucidate mechanisms responsible for the antiglioma effects of LY317615, we performed gene expression profiling in hopes of identifying potential downstream effectors of PKC-b inhibition. Striking, were alterations among components of the Wnt pathway within the nearly 1400 mRNA transcripts significantly altered following glioma cell exposure to LY317615. The strongest up-regulated gene (more than 40-fold) was axin 2 mRNA, a known component of the Wnt negative feedback loop. In addition to axin 2, we found highly significant changes in expression of at least 20 genes, such as CyclinD1, that are known to be the targets of b-catenin, the down-stream effector of the Wnt pathway. Further investigation of this pathway by both pharmacological and genetic means have suggested that activation of Wnt pathway in glioma cell lines leads to cell death. Specifically, we have demonstrated that the potency of the cytotoxic effects is directly correlated with decreased enzyme activity-activating phosphorylation of GSK3 alpha/beta Y276/Y216 and with increased enzyme activity-inhibitory phosphorylation of GSK3alpha S21. Inhibition of GSK3 activity results in a cytotoxicity-dependent increase in c-MYC activity thereby inducing expression of Bim, bax and DR4/DR5. Down-regulation of GSK3 activity also leads to a drop in FLIP protein and up-regulation of TRAIL. In addition to up-regulation of components of the TRAIL-associated extrinsic apoptotic pathway, downregulation of GSK3 activity results in alteration of intracellular glucose metabolism resulting in dissociation of hexokinase (HK) II from outer mitochondrial membrane with subsequent mitochondrial destabilization. Finally, inhibition of GSK3 activity causes a dramatic decrease in intracellular nuclear factor-kappa B (NF--B) activity. Thus, inhibition of GSK3 activity results in c-MYC dependent glioma cell death through multiple mechanisms all of which converge on the apoptotic pathways. These data support the hypothesis that GSK3 may be important therapeutic target for gliomas. Based on the promising preclinical data, we initiated a clinical trial of LY317615 in patients with recurrent high-grade gliomas