Elucidation and Exploitation of GSK3 as a Novel Glioma Therapeutic Target

GSK3 作为新型神经胶质瘤治疗靶点的阐明和开发

• 批准号: 8552857
• 负责人: Howard Fine
• 金额: $ 54.26万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552857
• 关键词: AXIN2 protein; Aftercare; Apoptosis; Apoptotic; Bromodeoxyuridine; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell Proliferation; Cell Survival; Cell physiology; Cells; Clinical Trials; Data; Dissociation; Down-Regulation; Drops; Employee Strikes; Exposure to; Feedback; G2/M Arrest; Gene Expression Profiling; Genes; Genetic; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Hexokinase 2; Inhibitory Concentration 50; Investigation; Isoenzymes; LY317615; Mediating; Messenger RNA; Metabolism; Mitochondria; Modeling; Molecular; NF-kappa B; Organism; Outer Mitochondrial Membrane; PRKCB1 gene; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phosphorylation; Protein-Serine-Threonine Kinases; Proteins; Recurrence; Role; Staining method; Stains; Stream; TNFRSF10A gene; TNFRSF10B gene; TNFSF10 gene; Therapeutic; Transcript; U251; Up-Regulation; Vascular Endothelial Growth Factors; angiogenesis; base; cytotoxic; cytotoxicity; enzyme activity; glioma cell line; glucose metabolism; inhibitor/antagonist; neoplastic cell; novel; pre-clinical; small molecule; therapeutic target; tumor

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

我们已经证明，GSK3 活性的多种小分子抑制剂和 GSK3/ 基因下调可显着抑制神经胶质瘤细胞的存活。在使用的小分子中，LY317615由礼来制药公司开发，作为ATP竞争性PKC-β（PKC-b）抑制剂，抑制VEGF刺激的内皮增殖，并应用于临床前肿瘤模型，显示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 活性直接对神经胶质瘤细胞发挥抗增殖活性。我们发现 LY317615 在药理学可达到的浓度（IC50 为 10mM）下对神经胶质瘤细胞系发挥有效的抗增殖活性。我们试图确定 LY317615 对神经胶质瘤细胞的抗增殖机制。通过对 LY317615 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 小时就出现了药物诱导的 G2/M 期停滞和细胞凋亡。为了阐明 LY317615 抗神经胶质瘤作用的机制，我们进行了基因表达谱分析，希望能够识别 PKC-b 抑制的潜在下游效应子。引人注目的是，神经胶质瘤细胞暴露于 LY317615 后，近 1400 个 mRNA 转录本中的 Wnt 通路成分发生了显着改变。上调最强的基因（超过 40 倍）是 axin 2 mRNA，它是 Wnt 负反馈环的已知组成部分。除了axin 2之外，我们还发现至少20个基因（例如CyclinD1）的表达发生了高度显着的变化，这些基因已知是b-连环蛋白（Wnt途径的下游效应子）的靶标。通过药理学和遗传学手段对该途径的进一步研究表明，神经胶质瘤细胞系中 Wnt 途径的激活会导致细胞死亡。具体来说，我们已经证明细胞毒性作用的效力与GSK3/Y276/Y216的酶活性激活磷酸化的降低和GSK3 S21的酶活性抑制磷酸化的增加直接相关。 GSK3 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 DR4/DR5 的表达。 GSK3 活性的下调也会导致 FLIP 蛋白的下降和 TRAIL 的上调。除了 TRAIL 相关的外源性细胞凋亡途径的成分上调外，GSK3 活性的下调还会导致细胞内葡萄糖代谢的改变，从而导致己糖激酶 (HK) II 从线粒体外膜解离，从而导致线粒体不稳定。最后，抑制 GSK3 活性会导致细胞内核因子-κ B (NF-) 活性急剧下降。因此，抑制 GSK3 活性会通过多种机制导致 c-MYC 依赖性神经胶质瘤细胞死亡，所有这些机制都集中在细胞凋亡途径上。这些数据支持 GSK3 可能是神经胶质瘤的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 LY317615 的临床试验

项目成果

GliomaPredict: a clinically useful tool for assigning glioma patients to specific molecular subtypes.

• DOI: 10.1186/1472-6947-10-38
• 发表时间: 2010-07-15
• 期刊: BMC medical informatics and decision making
• 影响因子: 3.5
• 作者: Li A;Bozdag S;Kotliarov Y;Fine HA
• 通讯作者: Fine HA

Malignant gliomas: new translational therapies.

• DOI: 10.1002/msj.20223
• 发表时间: 2010-11
• 期刊: The Mount Sinai journal of medicine, New York
• 作者: Sul J;Fine HA
• 通讯作者: Fine HA

Integration and analysis of genome-scale data from gliomas.

• DOI: 10.1038/nrneurol.2011.100
• 发表时间: 2011-07-05
• 期刊: Nature reviews. Neurology
• 作者: Riddick G;Fine HA
• 通讯作者: Fine HA

Correlation analysis between single-nucleotide polymorphism and expression arrays in gliomas identifies potentially relevant target genes.

• DOI: 10.1158/0008-5472.can-08-2496
• 发表时间: 2009-02-15
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Kotliarov Y;Kotliarova S;Charong N;Li A;Walling J;Aquilanti E;Ahn S;Steed ME;Su Q;Center A;Zenklusen JC;Fine HA
• 通讯作者: Fine HA

Unsupervised analysis of transcriptomic profiles reveals six glioma subtypes.

• DOI: 10.1158/0008-5472.can-08-2100
• 发表时间: 2009-03-01
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Li A;Walling J;Ahn S;Kotliarov Y;Su Q;Quezado M;Oberholtzer JC;Park J;Zenklusen JC;Fine HA
• 通讯作者: Fine HA