Elucidation and Exploitation of GSK3 as a Novel Glioma Therapeutic Target

我们已经证明，GSK3活性的多个小分子抑制剂和GSK3/基因下调显著抑制胶质瘤细胞的存活。在使用的小分子中，LY317615是由礼来制药公司开发的pkc - β (PKC-b)的atp竞争性抑制剂，可抑制vegf刺激的内皮细胞增殖，并应用于临床前肿瘤模型，显示出显著的抗血管生成活性。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Enzastaurin是否可以通过抑制PKC-b活性直接对胶质瘤细胞发挥抗增殖活性。我们发现LY317615在药理学上可达到的浓度（IC50为10mM）下对胶质瘤细胞系具有有效的抗增殖活性。我们试图确定LY317615对胶质瘤细胞的抗增殖机制。通过BrdU/PI染色对ly317615处理的U251进行细胞周期分析，发现药物诱导的G2/M阻滞和凋亡早在处理后24小时发生。为了阐明LY317615抗胶质瘤作用的机制，我们进行了基因表达谱分析，希望找出PKC-b抑制的潜在下游效应物。令人震惊的是，在胶质瘤细胞暴露于LY317615后，近1400个mRNA转录本中Wnt通路组分的改变显著改变。上调幅度最大的基因是轴蛋白2 mRNA（超过40倍），这是Wnt负反馈回路的已知成分。除了轴蛋白2外，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CyclinD1，这些基因已知是b-catenin （Wnt通路的下游效应物）的靶标。通过药理学和遗传学手段对该通路的进一步研究表明，胶质瘤细胞系中Wnt通路的激活可导致细胞死亡。具体来说，我们已经证明了细胞毒性作用的效力与GSK3 / Y276/Y216酶活性的降低（激活磷酸化）和GSK3 S21酶活性的增加（抑制磷酸化）直接相关。抑制GSK3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DR4/DR5的表达。GSK3活性的下调也导致FLIP蛋白的下降和TRAIL的上调。除了上调trail相关的外源性凋亡通路成分外，GSK3活性的下调还会导致细胞内葡萄糖代谢的改变，导致己糖激酶（HK） II从线粒体外膜分离，从而导致线粒体不稳定。最后，抑制GSK3活性导致胞内核因子- κ B （NF-）活性显著降低。因此，抑制GSK3活性通过多种机制导致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。这些数据支持了GSK3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，我们启动了LY317615在复发性高级别胶质瘤患者中的临床试验

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