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]

转化科学神经肿瘤分支实验室分为三个主要领域： 项目 3：糖原合酶激酶 3b (GSK3-b) 抑制的潜在治疗作用，从而增加 β-连环蛋白活性，控制神经胶质瘤介导的血管生成和神经胶质瘤细胞增殖：糖原合成酶激酶 3 (GSK3) 是一种存在于所有真核生物中的丝氨酸/苏氨酸激酶，参与细胞代谢等多种细胞功能对细胞周期的调节。我们已经证明，GSK3 活性的多种小分子抑制剂和 GSK3α/β 的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 α/β Y276/Y216 的酶活性激活磷酸化的减少和 GSK3α 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-κB) 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。 干细胞因子（SCF）作为一种重要的新型胶质瘤，在高级别胶质瘤中产生和神经元诱导的血管生成因子。 SCF 的表达过去已在许多神经胶质瘤细胞系中得到证实，但其意义仍不清楚，因为 SCF 在体外似乎对神经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检测到 SCF 表达，但我们发现与非肿瘤大脑相比，它在神经胶质瘤细胞系和神经胶质瘤中都以高水平表达。此外，与低级别神经胶质瘤相比，高级别神经胶质瘤中的 SCF 表达水平具有统计学显着更高的水平。由于与低级别神经胶质瘤相比，高级别神经胶质瘤的特点是肿瘤相关血管生成量大得多，因此 SCF 表达与神经胶质瘤级别增加的正相关性与 SCF 在神经胶质瘤相关血管生成中的潜在作用一致。我们已经证明，SCF 受体 c-Kit 在所检查的所有内皮细胞 (EC) 的表面上表达，并且将基础培养基中的 BMVEC-b、HUVEC 和 HMVEC-d 暴露于 SCF 会导致胸苷掺入和细胞增殖在所有 3 个 EC 系中，即使在没有其他细胞因子（如 VEGF）的情况下，即使在低浓度下，也以剂量依赖的方式。 SCF还在体外伤口愈合试验和毛细管形成试验中诱导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，我们将浸渍有 SCF、b-FGF（阳性对照）或单独载体的基质胶植入成年 SCID 小鼠体内。获得的数据表明SCF可以促进体内血管生成。通过类似的技术，我们还证明了抑制神经胶质瘤细胞中的 SCF 会显着抑制神经胶质瘤诱导的体内血管生成。接下来我们评估了抑制 SCF 是否会影响患有颅内神经胶质瘤的动物的生存。 U373/as-SCF 或 U373/vector 细胞被立体定向​​植入成年无胸腺 nu/nu 小鼠的大脑皮层下。 Kaplan-Meier 生存曲线的对数秩分析表明，与 U373/载体对照小鼠相比，U373/as-SCF 小鼠具有显着的生存优势 (P<0.05)，尽管两种细胞的生长速率均不同。体外类型相同。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的免疫组织化学分析揭示了继发于肿瘤和神经元相关 SCF 表达的神经胶质瘤细胞浸润的大脑皮层中 SCF 的深度表达。总之，SCF 表达似乎最突出地存在于浸润性神经胶质瘤的侵袭性前沿，表明其在肿瘤进展中的作用。鉴于我们的数据证明了 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但主要的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，我们开展了一系列体内实验，以研究格列卫对原位神经胶质瘤模型中神经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，评估格列卫与特定VEGF抑制剂（LY317615、Avastin等）的联合用药。然而，格列卫对完整血脑屏障的渗透性较差，也将迫使我们筛选其他药物[摘要被截断为 7800 个字符]