Elucidation and Exploitation of GSK3 as a Novel Glioma Therapeutic Target

我们已经证明，多种小分子抑制剂的GSK 3活性和基因下调的GSK 3/显着抑制胶质瘤细胞的存活。在所使用的小分子中，LY 317615由Eli Lilly Pharmaceuticals开发，作为PKC-β（PKC-b）的ATP竞争性抑制剂，以抑制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。考虑到其他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活性直接对神经胶质瘤细胞发挥抗增殖活性。我们发现LY 317615在可达到的浓度（IC 50为10 mM）下对胶质瘤细胞系发挥有效的抗增殖活性。我们试图确定LY 317615对胶质瘤细胞的抗增殖机制。通过对LY 317615处理的U251进行BrdU/PI染色进行的细胞周期分析显示，早在处理后24小时，药物就诱导了G2/M期阻滞和细胞凋亡。为了阐明LY 317615抗胶质瘤作用的机制，我们进行了基因表达谱分析，希望确定潜在的下游效应的PKC-b抑制。引人注目的是，在神经胶质瘤细胞暴露于LY 317615后，近1400种mRNA转录物中Wnt途径组分之间的改变显著改变。最强的上调基因（超过40倍）是axin 2 mRNA，Wnt负反馈环的一个已知成分。除了轴蛋白2，我们还发现至少20个基因的表达发生了高度显著的变化，如CyclinD 1，这些基因是已知的Wnt通路下游效应子β-连环蛋白的靶基因。通过药理学和遗传学手段对该通路的进一步研究表明，在胶质瘤细胞系中，Wnt通路的激活导致细胞死亡。具体而言，我们已经证明细胞毒性作用的效力与GSK 3/ Y276/Y216的酶活性激活磷酸化降低和GSK 3 S21的酶活性抑制磷酸化增加直接相关。GSK 3活性的抑制导致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蛋白的下降和TRAIL的上调。除了上调TRAIL相关的外源性凋亡途径的组分外，下调GSK 3活性导致细胞内葡萄糖代谢的改变，导致己糖激酶（HK）II从线粒体外膜解离，随后线粒体失稳。最后，抑制GSK 3活性导致细胞内核因子-κ B（NF-）活性显著降低。因此，GSK 3活性的抑制通过多种机制导致c-MYC依赖性胶质瘤细胞死亡，所有这些机制都集中在凋亡途径上。这些数据支持GSK 3可能是胶质瘤重要治疗靶点的假设。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 GSK3/ 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 / Y276/Y216 and with increased enzyme activity-inhibitory phosphorylation of GSK3 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-) 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