The Pre-clinical and Clinical Development of Novel Molecularly Target

新型分子靶点的临床前和临床开发

• 批准号: 7592987
• 负责人: Howard Fine
• 金额: $ 236.86万
• 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/7592987
• 关键词: AXIN2 protein; Adult; Aftercare; Angiogenesis Inhibitors; Angiogenic Factor; Animals; Apoptosis; Apoptotic; Area; Avastin; Bioinformatics; Biological Assay; Blood - brain barrier anatomy; Blood capillaries; Brain Neoplasms; Bromodeoxyuridine; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cell physiology; Cells; Cellular biology; Cerebral cortex; Cerebrum; Clinical; Clinical Trials; Condition; Data; Databases; Development; Diagnostic; Dissociation; Dose; Down-Regulation; Drops; Employee Strikes; Endothelial Cells; Exposure to; Facility Construction Funding Category; Feedback; G2/M Arrest; Gene Expression Profiling; Genes; Genetic; Gleevec; Glioblastoma; Glioma; Gliomagenesis; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Growth; Hexokinase 2; Human; Implant; In Vitro; Inhibitory Concentration 50; Invasive; Investigation; Isoenzymes; LY317615; Laboratories; Mediating; Messenger RNA; Metabolism; Mitochondria; Modeling; Molecular; Molecular Genetics; Molecular Target; Mus; NF-kappa B; Numbers; Operative Surgical Procedures; Organism; Outer Mitochondrial Membrane; PRKCB1 gene; Pathway interactions; Patients; Penetration; Pharmaceutical Preparations; Pharmacologic Substance; Phosphorylation; Protein-Serine-Threonine Kinases; Proteins; Proto-Oncogene Protein c-kit; Purpose; Range; Rate; Recurrence; Role; SCID Mice; Sampling; Secondary to; Series; Signal Transduction; Specimen; Staining method; Stains; Statistically Significant; Stem Cell Factor; Stem cells; Stream; Subgroup; Surface; TNFSF10 gene; Taxonomy; Technology; Therapeutic; Therapeutic Agents; Thymidine; Transcript; Transcriptional Activation; Translational Research; Tube; U251; Up-Regulation; Validation; Vascular Endothelial Growth Factors; Wound Healing; angiogenesis; base; capillary; cell motility; cell type; cytokine; cytotoxic; cytotoxicity; drug discovery; enzyme activity; glucose metabolism; in vivo; inhibitor/antagonist; matrigel; migration; neoplastic cell; nerve stem cell; neuro-oncology; neuronal tumor; novel; pre-clinical; preclinical study; protein kinase C beta; research study; small molecule; therapeutic target; tool; tumor; tumor progression; vector; vector control

The Neuro-Oncology Branch Laboratory of translational science is divided into three major areas: Project 3: The potential therapeutic role of Glycogen Synthase kinase-3b (GSK3-b) inhibition with resultant increases in b-catenin activity in controlling glioma-mediated angiogenesis and glioma cell proliferation: Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase found in all eukaryotic organisms and is involved in multiple cellular functions ranging from cellular metabolism to cell cycle regulation. We have demonstrated that multiple small molecular inhibitors of GSK3 activity and genetic downregulation of GSK3&#945;/&#946; 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 &#945;/&#946; Y276/Y216 and with increased enzyme activity-inhibitory phosphorylation of GSK3&#945; 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-&#954;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. Stem Cell Factor (SCF) as an important new glioma produced and neuronally induced angiogenic factor in high-grade gliomas. SCF expression has been demonstrated in the past in a number of glioma cell lines although its significance remains unclear since SCF does not appear to have any direct effects on glioma cells in vitro. Under normal conditions, little or no SCF expression is detectable in normal cerebrum, however we found it to be expressed at high levels both in glioma cells lines and in gliomas when compared to non-tumor brain. Additionally, there was a statistically significant higher level of SCF expression in high-grade gliomas compared to low-grade gliomas. Since high-grade gliomas are characterized by a much greater amount of tumor-associated angiogenesis compared to low-grade gliomas, the positive correlation of SCF expression with increasing glioma grade is consistent with a potential role for SCF in glioma-associated angiogenesis. We have demonstrated that the SCF receptor, c-Kit, is expressed on the surface of all endothelial cells (ECs) examined and that exposure of BMVEC-b, HUVEC and HMVEC-d in basal medium to SCF resulted in thymidine incorporation and cellular proliferation in all 3 EC lines in a dose-dependent manner even at low concentrations in the absence of other cytokines such as VEGF. SCF also induced EC migration and differentiation in an in vitro wound healing assay and capillary tube formation assay. These data demonstrated the ability of SCF to induce proliferation, migration and differentiation of BMVEC-b in vitro. We next subcutaneously implanted Matrigel impregnated with SCF, b-FGF (positive control) or vehicle alone into the adult SCID mice. The data obtained demonstrated that SCF can promote angiogenesis in vivo. By a similar technology we also demonstrated that suppression of SCF in glioma cells results in significant inhibition of glioma-induced angiogenesis in vivo. We next evaluated whether suppression of SCF would effect the survival of animals with intracranial gliomas. U373/as-SCF or U373/vector cells were stereotactically implanted to the cerebral subcortex of adult athymic nu/nu mice. Log-rank analysis of the Kaplan-Meier survival curves demonstrated a significant survival advantage for the U373/as-SCF bearing mice compared to the U373/vector control bearing animals (P<0.05), despite the fact that the growth rate of both cells types in vitro was identical. To confirm these results in actual tumor samples, immunohistochemical analysis of multiple surgical specimens from patients with glioblastoma revealed profound expression of SCF in cerebral cortex infiltrated by glioma cells secondary to both tumor-and neuronal-associated SCF expression. In summary, SCF expression appears to reside most prominently in the invasive front of the infiltrating glioma, suggesting its roles in the tumor progression. Given our data demonstrating the importance of SCF in tumor and host cell-induced angiogenesis, we hypothesize that a previously unrecognized, but major anti-tumor mechanism of Gleevec may be as an anti-angiogenic agent through its ability to potently inhibit c-kit signaling. We have therefore embarked on a series of in vivo experiments to look at the effects of Gleevec on glioma-mediated angiogenesis in our orthotopic glioma models. Thus, we will embark on a series of preclinical studies evaluating the combination of Gleevec with specific VEGF inhibitors (LY317615, Avastin, etc.). The poor penetration of Gleevec through an intact blood-brain barrier, however, will also force us to screen oth [summary truncated at 7800 characters]

转化科学神经肿瘤学分支实验室分为三个主要领域：项目3：糖原合成酶激酶3b （GSK3-b）抑制与由此引起的b-连环蛋白活性增加在控制胶质瘤介导的血管生成和胶质瘤细胞增殖中的潜在治疗作用；糖原合成酶激酶-3 （GSK3）是一种丝氨酸/苏氨酸激酶，存在于所有真核生物中，参与多种细胞功能，从细胞代谢到细胞周期调节。我们已经证明，GSK3活性的多种小分子抑制剂和GSK3&#945；/&#946；的遗传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降低酶活性-激活GSK3&#945； /&#946； Y276/Y216和增加酶活性-抑制GSK3&#945； S21的磷酸化直接相关。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-&#954；B）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。干细胞因子（Stem Cell Factor， SCF）是高级别胶质瘤产生和神经诱导的重要新生血管生成因子。过去在许多胶质瘤细胞系中已经证实了SCF的表达，尽管其意义尚不清楚，因为SCF在体外似乎对胶质瘤细胞没有任何直接影响。在正常情况下，正常大脑中很少或没有SCF表达，然而我们发现与非肿瘤大脑相比，它在胶质瘤细胞系和胶质瘤中都有高水平表达。此外，与低级别胶质瘤相比，高级别胶质瘤中SCF的表达水平有统计学意义。由于与低级别胶质瘤相比，高级别胶质瘤的特点是肿瘤相关血管生成的数量要大得多，因此SCF表达与胶质瘤级别的增加呈正相关，这与SCF在胶质瘤相关血管生成中的潜在作用是一致的。我们已经证明，SCF受体c-Kit在所有内皮细胞（ECs）表面表达，并且在基础培养基中BMVEC-b、HUVEC和HMVEC-d暴露于SCF导致胸苷结合和细胞增殖，即使在缺乏其他细胞因子（如VEGF）的低浓度下，也以剂量依赖的方式出现在所有3种EC系中。在体外伤口愈合实验和毛细管形成实验中，SCF还能诱导EC迁移和分化。这些数据证明了SCF在体外诱导BMVEC-b增殖、迁移和分化的能力。接下来，我们将单独浸渍SCF、b-FGF（阳性对照）或载药的Matrigel皮下植入成年SCID小鼠。实验结果表明，SCF能够促进体内血管生成。通过类似的技术，我们也证明了抑制胶质瘤细胞中的SCF可以显著抑制胶质瘤诱导的血管生成。我们接下来评估抑制SCF是否会影响颅内胶质瘤动物的存活。将U373/as-SCF或U373/载体细胞立体定向植入成年胸腺nu/nu小鼠的大脑皮层下。Kaplan-Meier生存曲线的Log-rank分析显示，携带U373/as-SCF的小鼠比携带U373/病媒对照的小鼠具有显著的生存优势（P<0.05），尽管两种细胞类型在体外的生长速度相同。为了在实际肿瘤样本中证实这些结果，对胶质母细胞瘤患者的多个手术标本进行免疫组织化学分析显示，在继发于肿瘤和神经元相关的SCF表达的胶质瘤细胞浸润的大脑皮层中，SCF的深度表达。总之，SCF表达似乎在浸润性胶质瘤的侵袭性前部最为显著，表明其在肿瘤进展中的作用。鉴于我们的数据表明SCF在肿瘤和宿主细胞诱导的血管生成中的重要性，我们假设格列卫以前未被认识到的主要抗肿瘤机制可能是通过其有效抑制c-kit信号传导的能力作为抗血管生成剂。因此，我们开始了一系列的体内实验，以观察格列卫对胶质瘤介导的血管生成的影响。因此，我们将开展一系列临床前研究，评估格列卫与特异性VEGF抑制剂（LY317615、阿瓦斯汀等）的联合应用。然而，格列卫通过完整血脑屏障的渗透性差，也将迫使我们筛选其他[摘要删节为7800个字符]。