Elucidation and Exploitation of GSK3 as a Novel Glioma Therapeutic Target

我们已经证明，GSK3活性的多种小分子抑制剂和GSK3α/β基因下调显著抑制胶质瘤细胞的存活。在所使用的小分子中，LY317615是由礼来制药公司开发的一种ATP竞争性PKC-β抑制剂(PKC-b)，用于抑制血管内皮生长因子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同工酶已被证明对肿瘤细胞的存活和增殖有贡献，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在药理上可达到的浓度(IC50为10 mM)对胶质瘤细胞株具有很强的抗增殖活性。本研究旨在探讨LY317615抗胶质瘤细胞增殖的作用机制。LY317615处理的U251细胞经BrdU/PI染色后进行细胞周期分析，发现药物作用后24小时即出现G2/M期阻滞和细胞凋亡。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，希望确定PKC-b抑制的潜在下游效应。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途径的组成部分发生了显著变化。上调最强的基因(超过40倍)是Axin 2mRNA，它是Wnt负反馈环的已知组成部分。除了Axin 2外，我们还发现至少20个基因的表达发生了非常显著的变化，例如CyclinD1，这些基因是已知的b-catenin的靶标，b-catenin是Wnt途径的下游效应因子。进一步的药理学和遗传学研究表明，在胶质瘤细胞系中，Wnt通路的激活导致细胞死亡。具体地说，我们已经证明，细胞毒作用的效力与酶活性的降低直接相关-激活GSK3α/βY276/Y216的磷酸化，而增加酶活性-抑制GSK3αS21的磷酸化。抑制GSK3活性导致c-myc活性依赖于细胞毒性增加，从而诱导Bim、Bax和DR4/DR5的表达。GSK3活性的下调也会导致翻转蛋白的下降和TRAIL的上调。除了上调TRAIL相关的外源性凋亡途径的组成部分外，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从线粒体膜外解离，从而导致线粒体失稳。最后，GSK3活性的抑制会导致细胞内核因子-kappaB(NF-kappaB)活性的急剧下降。因此，抑制GSK3活性通过多种机制导致c-myc依赖的胶质瘤细胞死亡，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在复发的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 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-kappaB) 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